Car enzymes and improved production of fatty alcohols

ABSTRACT

The disclosure relates to variant carboxylic acid reductase (CAR) enzymes for the improved production of fatty alcohols in recombinant host cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/390,350, filed Oct. 2, 2014, which is the National Stage ofInternational Application No. PCT/US2013/035040, filed Apr. 2, 2013,which claims the benefit of U.S. Provisional Application No. 61/619,309,filed Apr. 2, 2012, the entire disclosures of which are herebyincorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing, which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. The ASCII copy, created on Apr. 2, 2013, isnamed LS00039PCT_SL.txt and is 89,038 bytes in size.

FIELD OF THE DISCLOSURE

The disclosure relates to variant carboxylic acid reductase (CAR)enzymes for the improved production of fatty alcohols in recombinanthost cells. The disclosure further relates to variant CAR nucleic acidsand polypeptides as well as recombinant host cells and cell cultures.Further encompassed are methods of making fatty alcohol compositions.

BACKGROUND OF THE DISCLOSURE

Fatty alcohols make up an important category of industrial biochemicals.These molecules and their derivatives have numerous uses, including assurfactants, lubricants, plasticizers, solvents, emulsifiers,emollients, thickeners, flavors, fragrances, and fuels. In industry,fatty alcohols are produced via catalytic hydrogenation of fatty acidsproduced from natural sources, such as coconut oil, palm oil, palmkernel oil, tallow and lard, or by chemical hydration of alpha-olefinsproduced from petrochemical feedstock. Fatty alcohols derived fromnatural sources have varying chain lengths. The chain length of fattyalcohols is important with respect to particular applications. Innature, fatty alcohols are also made by enzymes that are able to reduceacyl-ACP or acyl-CoA molecules to the corresponding primary alcohols(see, for example, U.S. Patent Publication Nos. 20100105955,20100105963, and 20110250663, which are incorporated by referenceherein).

Current technologies for producing fatty alcohols involve inorganiccatalyst-mediated reduction of fatty acids to the corresponding primaryalcohols, which is costly, time consuming and cumbersome. The fattyacids used in this process are derived from natural sources (e.g., plantand animal oils and fats, supra). Dehydration of fatty alcohols toalpha-olefins can also be accomplished by chemical catalysis. However,this technique is nonrenewable and associated with high operating costand environmentally hazardous chemical wastes. Thus, there is a need forimproved methods for fatty alcohol production and the instant disclosureaddresses this need.

SUMMARY

One aspect of the disclosure provides a variant carboxylic acidreductase (CAR) polypeptide comprising an amino acid sequence having atleast about 90% sequence identity to SEQ ID NO: 7, wherein the variantCAR polypeptide is genetically engineered to have at least one mutationat an amino acid position selected from the group of amino acidpositions 3, 18, 20, 22, 80, 87, 191, 288, 473, 535, 750, 827, 870, 873,926, 927, 930, and 1128. Herein, the expression of the variant CARpolypeptide in a recombinant host cell results in a higher titer offatty alcohol compositions compared to a recombinant host cellexpressing a corresponding wild type polypeptide. In a related aspect,the CAR polypeptide is a CarB polypeptide. In another related aspect,the variant CAR polypeptide comprises a mutation at positions S3R, D18R,D18L, D18T, D18P, E20V, E20S, E20R, S22R, S22N, S22G, L80R, R87G, R87E,V191S, F288R, F288S, F288G, Q473L, Q473W, Q473Y, Q473I, Q473H, A535S,D750A, R827C, R827A, I870L, R873S, V926A, V926E, S927K, S927G, M930K,M930R and/or L1128W. In a related aspect, the CAR polypeptide includesmutation A535S; or mutations E20R, F288G, Q473I and A535S; or mutationsE20R, F288G, Q473H, A535S, R827A and S927G; or mutations E20R, S22R,F288G, Q473H, A535S, R827A and S927G; or mutations S3R, E20R, S22R,F288G, Q473H, A535S, R873S, S927G, M930R and L1128W; or E20R, S22R,F288G, Q473H, A535S, R873S, S927G, M930R and L1128W; or mutations D18R,E20R, S22R, F288G, Q473I, A535S, S927G, M930K and L1128W; or mutationsE20R, S22R, F288G, Q473I, A535S, R827C, V926E, S927K and M930R; ormutations D18R, E20R, 288G, Q473I, A535S, R827C, V926E, M930K andL1128W; or mutations E20R, S22R, F288G, Q473H, A535S, R827C, V926A,S927K and M930R; or mutations E20R, S22R, F288G, Q473H, A535S and R827C;or mutations E20R, S22R, F288G, Q473I, A535S, R827C and M930R; ormutations E20R, S22R, F288G, Q473I, A535S, I870L, S927G and M930R; ormutations E20R, S22R, F288G, Q473I, A535S, I870L and S927G; or mutationsD18R, E20R, S22R, F288G, Q473I, A535S, R827C, I870L, V926A and S927G; ormutations E20R, S22R, F288G, Q473H, A535S, R827C, I870L and L1128W; ormutations D18R, E20R, S22R, F288G, Q473H, A535S, R827C, I870L, S927G andL1128W; or mutations E20R, S22R, F288G, Q473I, A535S, R827C, I870L,S927G and L1128W; or mutations E20R, S22R, F288G, Q473I, A535S, R827C,I870L, S927G, M930K and L1128W; or mutations E20R, S22R, F288G, Q473H,A535S, I870L, S927G and M930K; or mutations E20R, F288G, Q473I, A535S,I870L, M930K; or mutations E20R, S22R, F288G, Q473H, A535S, S927G, M930Kand L1128W; or mutations D18R, E20R, S22R, F288G, Q473I, A535S, S927Gand L1128W; or mutations E20R, S22R, F288G, Q473I, A535S, R827C, I870Land S927G; or mutations D18R, E20R, S22R, F288G, Q473I, A535S, R827C,I870L, S927G and L1128W; or mutations D18R, E20R, S22R, F288G, Q473I,A535S, S927G, M930R and L1128W; or mutations E20R, S22R, F288G, Q473H,A535S, V926E, S927G and M930R; or mutations E20R, S22R, F288G, Q473H,A535S, R827C, I870L, V926A and L1128W; or combinations thereof.

Another aspect of the disclosure provides a host cell including apolynucleotide sequence encoding a variant carboxylic acid reductase(CAR) polypeptide having at least 90% sequence identity to SEQ ID NO: 7and having at least one mutation at an amino acid position includingamino acid positions 3, 18, 20, 22, 80, 87, 191, 288, 473, 535, 750,827, 870, 873, 926, 927, 930, and 1128, wherein the geneticallyengineered host cell produces a fatty alcohol composition at a highertiter or yield than a host cell expressing a corresponding wild type CARpolypeptide when cultured in a medium containing a carbon source underconditions effective to express the variant CAR polypeptide, and whereinthe SEQ ID NO: 7 is the corresponding wild type CAR polypeptide. In arelated aspect, the recombinant host cell further includes apolynucleotide encoding a thioesterase polypeptide. In another relatedaspect, the recombinant host cell further includes a polynucleotideencoding a FabB polypeptide and a FadR polypeptide. In another relatedaspect, the disclosure provides a recombinant host cell that includes apolynucleotide encoding a fatty aldehyde reductase (AlrA) and a cellculture containing it.

Another aspect of the disclosure provides a recombinant host cell,wherein the genetically engineered host cell has a titer that is atleast 3 times greater than the titer of a host cell expressing thecorresponding wild type CAR polypeptide when cultured under the sameconditions as the genetically engineered host cell. In one relatedaspect, the genetically engineered host cell has a titer of from about30 g/L to about 250 g/L. In another related aspect, the geneticallyengineered host cell has a titer of from about 90 g/L to about 120 g/L.

Another aspect of the disclosure provides a recombinant host cell,wherein the genetically engineered host cell has a yield that is atleast 3 times greater than the yield of a host cell expressing thecorresponding wild type CAR polypeptide when cultured under the sameconditions as the genetically engineered host cell. In one relatedaspect, the genetically engineered host cell has a yield from about 10%to about 40%.

The disclosure further encompasses a cell culture including therecombinant host cell as described herein. In a related aspect, the cellculture has a productivity that is at least about 3 times greater thanthe productivity of a cell culture that expresses the corresponding wildtype CAR polypeptide. In another related aspect, the productivity rangesfrom about 0.7 mg/L/hr to about 3 g/L/hr. In another related aspect, theculture medium comprises a fatty alcohol composition. The fatty alcoholcomposition is produced extracellularly. The fatty alcohol compositionmay include one or more of a C6, C8, C10, C12, C13, C14, C15, C16, C17,or C18 fatty alcohol; or a C10:1, C12:1, C14:1, C16:1, or a C18:1unsaturated fatty alcohol. In another related aspect, the fatty alcoholcomposition comprises C12 and C14 fatty alcohols. In yet, anotherrelated aspect, the fatty alcohol composition comprises C12 and C14fatty alcohols at a ratio of about 3:1. In still another related aspect,the fatty alcohol composition encompasses unsaturated fatty alcohols. Inaddition, the fatty alcohol composition may include a fatty alcoholhaving a double bond at position 7 in the carbon chain between C7 and C8from the reduced end of the fatty alcohol. In another aspect, the fattyalcohol composition includes saturated fatty alcohols. In anotheraspect, the fatty alcohol composition includes branched chain fattyalcohols.

The disclosure further contemplates a method of making a fatty alcoholcomposition at a high titer, yield or productivity, including the stepsof engineering a recombinant host cell; culturing the recombinant hostcell in a medium including a carbon source; and optionally isolating thefatty alcohol composition from the medium

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure is best understood when read in conjunction withthe accompanying figures, which serve to illustrate the preferredembodiments. It is understood, however, that the disclosure is notlimited to the specific embodiments disclosed in the figures.

FIG. 1 is a schematic overview of an exemplary biosynthetic pathway foruse in production of acyl CoA as a precursor to fatty acid derivativesin a recombinant host cell. The cycle is initiated by condensation ofmalonyl-ACP and acetyl-CoA.

FIG. 2 is a schematic overview of an exemplary fatty acid biosyntheticcycle, where malonyl-ACP is produced by the transacylation ofmalonyl-CoA to malonyl-ACP (catalyzed by malonyl-CoA:ACP transacylase;fabD), then β-ketoacyl-ACP synthase III (fabH) initiates condensation ofmalonyl-ACP with acetyl-CoA. Elongation cycles begin with thecondensation of malonyl-ACP and an acyl-ACP catalyzed by β-ketoacyl-ACPsynthase I (fabB) and β-ketoacyl-ACP synthase II (fabF) to produce aβ-keto-acyl-ACP, then the β-keto-acyl-ACP is reduced by aNADPH-dependent β-ketoacyl-ACP reductase (fabG) to produce aβ-hydroxy-acyl-ACP, which is dehydrated to a trans-2-enoyl-acyl-ACP byβ-hydroxyacyl-ACP dehydratase (fabA or fabZ). FabA can also isomerizetrans-2-enoyl-acyl-ACP to cis-3-enoyl-acyl-ACP, which can bypass fabIand can used by fabB (typically for up to an aliphatic chain length ofC16) to produce β-keto-acyl-ACP. The final step in each cycle iscatalyzed by a NADH or NADHPH-dependent enoyl-ACP reductase (fabI) thatconverts trans-2-enoyl-acyl-ACP to acyl-ACP. In the methods describedherein, termination of fatty acid synthesis occurs by thioesteraseremoval of the acyl group from acyl-ACP to release free fatty acids(FFA). Thioesterases (e.g., tesA) hydrolyze thioester bonds, which occurbetween acyl chains and ACP through sulfhydryl bonds.

FIG. 3 illustrates the structure and function of the acetyl-CoAcarboxylase (accABCD) enzyme complex. Biotin carboxylase is encoded bythe accC gene, whereas biotin carboxyl carrier protein (BCCP) is encodedby the accB gene. The two subunits involved in carboxyltransferaseactivity are encoded by the accA and accD genes. The covalently boundbiotin of BCCP carries the carboxylate moiety. The birA gene (not shown)biotinylates holo-accB.

FIG. 4 presents a schematic overview of an exemplary biosyntheticpathway for production of fatty alcohol starting with acyl-ACP, wherethe production of fatty aldehyde is catalyzed by the enzymatic activityof acyl-ACP reductase (AAR) or thioesterase and carboxylic acidreductase (Car). The fatty aldehyde is converted to fatty alcohol byaldehyde reductase (also referred to as alcohol dehydrogenase). Thispathway does not include fatty acyl CoA synthetase (fadD).

FIG. 5 illustrates fatty acid derivative (Total Fatty Species)production by the MG1655 E. coli strain with the fadE gene attenuated(i.e., deleted) compared to fatty acid derivative production by E. coliMG1655. The data presented in FIG. 5 shows that attenuation of the fadEgene did not affect fatty acid derivative production.

FIGS. 6A and 6B show data for production of “Total Fatty Species” fromduplicate plate screens when plasmid pCL-WT TRC WT TesA was transformedinto each of the strains shown in the figures and a fermentation was runin FA2 media with 20 hours from induction to harvest at both 32° C.(FIG. 6A) and 37° C. (FIG. 6B).

FIGS. 7A and 7B provide a diagrammatic depiction of the iFAB138 locus,including a diagram of cat-loxP-T5 promoter integrated in front ofFAB138 (7A); and a diagram of iT5_138 (7B). The sequence of cat-loxP-T5promoter integrated in front of FAB138 with 50 base pair of homologyshown on each side of cat-loxP-T5 promoter region is provided as SEQ IDNO:1 and the sequence of the iT5_138 promoter region with 50 base pairhomology on each side is provided as SEQ ID NO: 2.

FIG. 8 shows the effect of correcting the rph and ilvG genes. EG149(rph− ilvg−) and V668 (EG149 rph+ ilvG+) were transformed with pCL-tesA(a pCL1920 plasmid containing P_(TRC)-'tesA) obtained from D191. Thefigure shows that correcting the rph and ilvG genes in the EG149 strainallows for a higher level of FFA production than in the V668 strainwhere the rph and ilvG genes were not corrected.

FIG. 9 is a diagrammatic depiction of a transposon cassette insertion inthe yijP gene of strain LC535 (transposon hit 68F11). Promoters internalto the transposon cassette are shown, and may have effects on adjacentgene expression.

FIG. 10 shows conversion of free fatty acids to fatty alcohols by CarB60in strain V324. The figures shows that cells expressing CarB60 from thechromosome (dark bars) convert a greater fraction of C12 and C14 freefatty acids into fatty alcohol compared to CarB (light bars).

FIG. 11 shows that cells expressing CarB60 from the chromosome convert agreater fraction of C12 and C14 free fatty acids into fatty alcoholcompared to CarB.

FIG. 12 shows fatty alcohol production following fermentation ofcombination library mutants.

FIG. 13 shows fatty alcohol production by carB variants in productionplasmid (carB1 and CarB2) following shake-flask fermentation.

FIG. 14 shows fatty alcohol production by single-copy integrated carBvariants (icarB1 icarB2, icarB3, and icarB4) following shake-flaskfermentation.

FIG. 15 shows results of dual-plasmid screening system for improved CarBvariants as validated by shake-flask fermentation.

FIG. 16 shows novel CarB variants for improved production of fattyalcohols in bioreactors.

DETAILED DESCRIPTION

General Overview

The present disclosure provides novel variant carboxylic acid reductase(CAR) enzymes as well as their nucleic acid and protein sequences.Further encompassed by the disclosure are recombinant host cells andcell cultures that include the variant CAR enzymes for the production offatty alcohols. In order for the production of fatty alcohols fromfermentable sugars or biomass to be commercially viable, the processmust be optimized for efficient conversion and recovery of product. Thepresent disclosure addresses this need by providing compositions andmethods for improved production of fatty alcohols using engineeredvariant enzymes and engineered recombinant host cells. The host cellsserve as biocatalysts resulting in high-titer production of fattyalcohols using fermentation processes. As such, the disclosure furtherprovides methods to create photosynthetic and heterotrophic host cellsthat produce fatty alcohols and alpha-olefins of specific chain lengthsdirectly such that catalytic conversion of purified fatty acids is notnecessary. This new method provides product quality and cost advantages.

More specifically, the production of a desired fatty alcohol compositionmay be enhanced by modifying the expression of one or more genesinvolved in a biosynthetic pathway for fatty alcohol production,degradation and/or secretion. The disclosure provides recombinant hostcells, which have been engineered to provide enhanced fatty alcoholbiosynthesis relative to non-engineered or native host cells (e.g.,strain improvements). The disclosure also provides polynucleotidesuseful in the recombinant host cells, methods, and compositions of thedisclosure. However it will be recognized that absolute sequenceidentity to such polynucleotides is not necessary. For example, changesin a particular polynucleotide sequence can be made and the encodedpolypeptide evaluated for activity. Such changes typically compriseconservative mutations and silent mutations (e.g., codon optimization).Modified or mutated polynucleotides (i.e., mutants) and encoded variantpolypeptides can be screened for a desired function, such as, animproved function compared to the parent polypeptide, including but notlimited to increased catalytic activity, increased stability, ordecreased inhibition (e.g., decreased feedback inhibition), usingmethods known in the art.

The disclosure identifies enzymatic activities involved in various steps(i.e., reactions) of the fatty acid biosynthetic pathways describedherein according to Enzyme Classification (EC) number, and providesexemplary polypeptides (i.e., enzymes) categorized by such EC numbers,and exemplary polynucleotides encoding such polypeptides. Such exemplarypolypeptides and polynucleotides, which are identified herein byAccession Numbers and/or Sequence Identifier Numbers (SEQ ID NOs), areuseful for engineering fatty acid pathways in parental host cells toobtain the recombinant host cells described herein. It is to beunderstood, however, that polypeptides and polynucleotides describedherein are exemplary and non-limiting. The sequences of homologues ofexemplary polypeptides described herein are available to those of skillin the art using databases (e.g., the Entrez databases provided by theNational Center for Biotechnology Information (NCBI), the ExPasydatabases provided by the Swiss Institute of Bioinformatics, the BRENDAdatabase provided by the Technical University of Braunschweig, and theKEGG database provided by the Bioinformatics Center of Kyoto Universityand University of Tokyo, all which are available on the World Wide Web).

A variety of host cells can be modified to contain a fatty alcoholbiosynthetic enzymes such as those described herein, resulting inrecombinant host cells suitable for the production of fatty alcoholcompositions. It is understood that a variety of cells can providesources of genetic material, including polynucleotide sequences thatencode polypeptides suitable for use in a recombinant host cell providedherein.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the disclosure pertains. Although other methods andmaterials similar, or equivalent, to those described herein can be usedin the practice of the present disclosure, the preferred materials andmethods are described herein. In describing and claiming the presentdisclosure, the following terminology will be used in accordance withthe definitions set out below.

Accession Numbers: Sequence Accession numbers throughout thisdescription were obtained from databases provided by the NCBI (NationalCenter for Biotechnology Information) maintained by the NationalInstitutes of Health, U.S.A. (which are identified herein as “NCBIAccession Numbers” or alternatively as “GenBank Accession Numbers”), andfrom the UniProt Knowledgebase (UniProtKB) and Swiss-Prot databasesprovided by the Swiss Institute of Bioinformatics (which are identifiedherein as “UniProtKB Accession Numbers”).

Enzyme Classification (EC) Numbers: EC numbers are established by theNomenclature Committee of the International Union of Biochemistry andMolecular Biology (IUBMB), description of which is available on theIUBMB Enzyme Nomenclature website on the World Wide Web. EC numbersclassify enzymes according to the reaction catalyzed.

As used herein, the term “nucleotide” refers to a monomeric unit of apolynucleotide that consists of a heterocyclic base, a sugar, and one ormore phosphate groups. The naturally occurring bases (guanine, (G),adenine, (A), cytosine, (C), thymine, (T), and uracil (U)) are typicallyderivatives of purine or pyrimidine, though it should be understood thatnaturally and non-naturally occurring base analogs are also included.The naturally occurring sugar is the pentose (five-carbon sugar)deoxyribose (which forms DNA) or ribose (which forms RNA), though itshould be understood that naturally and non-naturally occurring sugaranalogs are also included. Nucleic acids are typically linked viaphosphate bonds to form nucleic acids or polynucleotides, though manyother linkages are known in the art (e.g., phosphorothioates,boranophosphates, and the like).

As used herein, the term “polynucleotide” refers to a polymer ofribonucleotides (RNA) or deoxyribonucleotides (DNA), which can besingle-stranded or double-stranded and which can contain non-natural oraltered nucleotides. The terms “polynucleotide,” “nucleic acidsequence,” and “nucleotide sequence” are used interchangeably herein torefer to a polymeric form of nucleotides of any length, either RNA orDNA. These terms refer to the primary structure of the molecule, andthus include double- and single-stranded DNA, and double- andsingle-stranded RNA. The terms include, as equivalents, analogs ofeither RNA or DNA made from nucleotide analogs and modifiedpolynucleotides such as, though not limited to methylated and/or cappedpolynucleotides. The polynucleotide can be in any form, including butnot limited to, plasmid, viral, chromosomal, EST, cDNA, mRNA, and rRNA.

As used herein, the terms “polypeptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The term“recombinant polypeptide” refers to a polypeptide that is produced byrecombinant techniques, wherein generally DNA or RNA encoding theexpressed protein is inserted into a suitable expression vector that isin turn used to transform a host cell to produce the polypeptide.

As used herein, the terms “homolog,” and “homologous” refer to apolynucleotide or a polypeptide comprising a sequence that is at leastabout 50% identical to the corresponding polynucleotide or polypeptidesequence. Preferably homologous polynucleotides or polypeptides havepolynucleotide sequences or amino acid sequences that have at leastabout 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or at least about 99% homology to thecorresponding amino acid sequence or polynucleotide sequence. As usedherein the terms sequence “homology” and sequence “identity” are usedinterchangeably.

One of ordinary skill in the art is well aware of methods to determinehomology between two or more sequences. Briefly, calculations of“homology” between two sequences can be performed as follows. Thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second amino acid or nucleicacid sequence for optimal alignment and non-homologous sequences can bedisregarded for comparison purposes). In a preferred embodiment, thelength of a first sequence that is aligned for comparison purposes is atleast about 30%, preferably at least about 40%, more preferably at leastabout 50%, even more preferably at least about 60%, and even morepreferably at least about 70%, at least about 80%, at least about 90%,or about 100% of the length of a second sequence. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions of the first and second sequences are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent homology between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps and the length of each gap, that need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent homologybetween two sequences can be accomplished using a mathematicalalgorithm, such as BLAST (Altschul et al., J. Mol. Biol., 215(3):403-410 (1990)). The percent homology between two amino acid sequencesalso can be determined using the Needleman and Wunsch algorithm that hasbeen incorporated into the GAP program in the GCG software package,using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6(Needleman and Wunsch, J. Mol. Biol., 48: 444-453 (1970)). The percenthomology between two nucleotide sequences also can be determined usingthe GAP program in the GCG software package, using a NWSgapdna.CMPmatrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of1, 2, 3, 4, 5, or 6. One of ordinary skill in the art can performinitial homology calculations and adjust the algorithm parametersaccordingly. A preferred set of parameters (and the one that should beused if a practitioner is uncertain about which parameters should beapplied to determine if a molecule is within a homology limitation ofthe claims) are a Blossum 62 scoring matrix with a gap penalty of 12, agap extend penalty of 4, and a frameshift gap penalty of 5. Additionalmethods of sequence alignment are known in the biotechnology arts (see,e.g., Rosenberg, BMC Bioinformatics, 6: 278 (2005); Altschul, et al.,FEBS J., 272(20): 5101-5109 (2005)).

As used herein, the term “hybridizes under low stringency, mediumstringency, high stringency, or very high stringency conditions”describes conditions for hybridization and washing. Guidance forperforming hybridization reactions can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Aqueousand non-aqueous methods are described in that reference and eithermethod can be used. Specific hybridization conditions referred to hereinare as follows: 1) low stringency hybridization conditions—6× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by two washes in0.2×SSC, 0.1% SDS at least at 50° C. (the temperature of the washes canbe increased to 55° C. for low stringency conditions); 2) mediumstringency hybridization conditions—6×SSC at about 45° C., followed byone or more washes in 0.2×SSC, 0.1% SDS at 60° C.; 3) high stringencyhybridization conditions—6×SSC at about 45° C., followed by one or morewashes in 0.2×SSC, 0.1% SDS at 65° C.; and 4) very high stringencyhybridization conditions—0.5M sodium phosphate, 7% SDS at 65° C.,followed by one or more washes at 0.2×SSC, 1% SDS at 65° C. Very highstringency conditions (4) are the preferred conditions unless otherwisespecified.

An “endogenous” polypeptide refers to a polypeptide encoded by thegenome of the parental microbial cell (also termed “host cell”) fromwhich the recombinant cell is engineered (or “derived”).

An “exogenous” polypeptide refers to a polypeptide, which is not encodedby the genome of the parental microbial cell. A variant (i.e., mutant)polypeptide is an example of an exogenous polypeptide.

The term “heterologous” generally means derived from a different speciesor derived from a different organism. As used herein it refers to anucleotide sequence or a polypeptide sequence that is not naturallypresent in a particular organism. Heterologous expression means that aprotein or polypeptide is experimentally added to a cell that does notnormally express that protein. As such, heterologous refers to the factthat a transferred protein was initially derived from a different celltype or a different species then the recipient. For example, apolynucleotide sequence endogenous to a plant cell can be introducedinto a bacterial host cell by recombinant methods, and the plantpolynucleotide is then a heterologous polynucleotide in a recombinantbacterial host cell.

As used herein, the term “fragment” of a polypeptide refers to a shorterportion of a full-length polypeptide or protein ranging in size fromfour amino acid residues to the entire amino acid sequence minus oneamino acid residue. In certain embodiments of the disclosure, a fragmentrefers to the entire amino acid sequence of a domain of a polypeptide orprotein (e.g., a substrate binding domain or a catalytic domain).

As used herein, the term “mutagenesis” refers to a process by which thegenetic information of an organism is changed in a stable manner.Mutagenesis of a protein coding nucleic acid sequence produces a mutantprotein. Mutagenesis also refers to changes in non-coding nucleic acidsequences that result in modified protein activity.

As used herein, the term “gene” refers to nucleic acid sequencesencoding either an RNA product or a protein product, as well asoperably-linked nucleic acid sequences affecting the expression of theRNA or protein (e.g., such sequences include but are not limited topromoter or enhancer sequences) or operably-linked nucleic acidsequences encoding sequences that affect the expression of the RNA orprotein (e.g., such sequences include but are not limited to ribosomebinding sites or translational control sequences).

Expression control sequences are known in the art and include, forexample, promoters, enhancers, polyadenylation signals, transcriptionterminators, internal ribosome entry sites (IRES), and the like, thatprovide for the expression of the polynucleotide sequence in a hostcell. Expression control sequences interact specifically with cellularproteins involved in transcription (Maniatis et al., Science, 236:1237-1245 (1987)). Exemplary expression control sequences are describedin, for example, Goeddel, Gene Expression Technology: Methods inEnzymology, Vol. 185, Academic Press, San Diego, Calif. (1990).

In the methods of the disclosure, an expression control sequence isoperably linked to a polynucleotide sequence. By “operably linked” ismeant that a polynucleotide sequence and an expression controlsequence(s) are connected in such a way as to permit gene expressionwhen the appropriate molecules (e.g., transcriptional activatorproteins) are bound to the expression control sequence(s). Operablylinked promoters are located upstream of the selected polynucleotidesequence in terms of the direction of transcription and translation.Operably linked enhancers can be located upstream, within, or downstreamof the selected polynucleotide.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid, i.e., a polynucleotidesequence, to which it has been linked. One type of useful vector is anepisome (i.e., a nucleic acid capable of extra-chromosomal replication).Useful vectors are those capable of autonomous replication and/orexpression of nucleic acids to which they are linked. Vectors capable ofdirecting the expression of genes to which they are operatively linkedare referred to herein as “expression vectors.” In general, expressionvectors of utility in recombinant DNA techniques are often in the formof “plasmids,” which refer generally to circular double stranded DNAloops that, in their vector form, are not bound to the chromosome. Theterms “plasmid” and “vector” are used interchangeably herein, inasmuchas a plasmid is the most commonly used form of vector. However, alsoincluded are such other forms of expression vectors that serveequivalent functions and that become known in the art subsequentlyhereto. In some embodiments, the recombinant vector comprises at leastone sequence including (a) an expression control sequence operativelycoupled to the polynucleotide sequence; (b) a selection markeroperatively coupled to the polynucleotide sequence; (c) a markersequence operatively coupled to the polynucleotide sequence; (d) apurification moiety operatively coupled to the polynucleotide sequence;(e) a secretion sequence operatively coupled to the polynucleotidesequence; and (f) a targeting sequence operatively coupled to thepolynucleotide sequence. The expression vectors described herein includea polynucleotide sequence described herein in a form suitable forexpression of the polynucleotide sequence in a host cell. It will beappreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression of polypeptide desired,etc. The expression vectors described herein can be introduced into hostcells to produce polypeptides, including fusion polypeptides, encoded bythe polynucleotide sequences as described herein.

Expression of genes encoding polypeptides in prokaryotes, for example,E. coli, is most often carried out with vectors containing constitutiveor inducible promoters directing the expression of either fusion ornon-fusion polypeptides. Fusion vectors add a number of amino acids to apolypeptide encoded therein, usually to the amino- or carboxy-terminusof the recombinant polypeptide. Such fusion vectors typically serve oneor more of the following three purposes: (1) to increase expression ofthe recombinant polypeptide; (2) to increase the solubility of therecombinant polypeptide; and (3) to aid in the purification of therecombinant polypeptide by acting as a ligand in affinity purification.Often, in fusion expression vectors, a proteolytic cleavage site isintroduced at the junction of the fusion moiety and the recombinantpolypeptide. This enables separation of the recombinant polypeptide fromthe fusion moiety after purification of the fusion polypeptide. Incertain embodiments, a polynucleotide sequence of the disclosure isoperably linked to a promoter derived from bacteriophage T5. In certainembodiments, the host cell is a yeast cell, and the expression vector isa yeast expression vector. Examples of vectors for expression in yeastS. cerevisiae include pYepSec1 (Baldari et al., EMBO J., 6: 229-234(1987)), pMFa (Kurjan et al., Cell, 30: 933-943 (1982)), pJRY88 (Schultzet al., Gene, 54: 113-123 (1987)), pYES2 (Invitrogen Corp., San Diego,Calif.), and picZ (Invitrogen Corp., San Diego, Calif.). In otherembodiments, the host cell is an insect cell, and the expression vectoris a baculovirus expression vector. Baculovirus vectors available forexpression of proteins in cultured insect cells (e.g., Sf9 cells)include, for example, the pAc series (Smith et al., Mol. Cell Biol., 3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology, 170:31-39 (1989)). In yet another embodiment, the polynucleotide sequencesdescribed herein can be expressed in mammalian cells using a mammalianexpression vector. Other suitable expression systems for bothprokaryotic and eukaryotic cells are well known in the art; see, e.g.,Sambrook et al., “Molecular Cloning: A Laboratory Manual,” secondedition, Cold Spring Harbor Laboratory, (1989).

As used herein “Acyl-CoA” refers to an acyl thioester formed between thecarbonyl carbon of alkyl chain and the sulfhydryl group of the4′-phosphopantethionyl moiety of coenzyme A (CoA), which has the formulaR—C(O)S-CoA, where R is any alkyl group having at least 4 carbon atoms.

As used herein “acyl-ACP” refers to an acyl thioester formed between thecarbonyl carbon of alkyl chain and the sulfhydryl group of thephosphopantetheinyl moiety of an acyl carrier protein (ACP). Thephosphopantetheinyl moiety is post-translationally attached to aconserved serine residue on the ACP by the action of holo-acyl carrierprotein synthase (ACPS), a phosphopantetheinyl transferase. In someembodiments an acyl-ACP is an intermediate in the synthesis of fullysaturated acyl-ACPs. In other embodiments an acyl-ACP is an intermediatein the synthesis of unsaturated acyl-ACPs. In some embodiments, thecarbon chain will have about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 carbons. Each of theseacyl-ACPs are substrates for enzymes that convert them to fatty acidderivatives.

As used herein, the term “fatty acid or derivative thereof” means a“fatty acid” or a “fatty acid derivative.” The term “fatty acid” means acarboxylic acid having the formula RCOOH. R represents an aliphaticgroup, preferably an alkyl group. R can comprise between about 4 andabout 22 carbon atoms. Fatty acids can be saturated, monounsaturated, orpolyunsaturated. In a preferred embodiment, the fatty acid is made froma fatty acid biosynthetic pathway. The term “fatty acid derivative”means products made in part from the fatty acid biosynthetic pathway ofthe production host organism. “Fatty acid derivative” also includesproducts made in part from acyl-ACP or acyl-ACP derivatives. Exemplaryfatty acid derivatives include, for example, acyl-CoA, fatty aldehydes,short and long chain alcohols, hydrocarbons, and esters (e.g., waxes,fatty acid esters, or fatty esters).

As used herein, the term “fatty acid biosynthetic pathway” means abiosynthetic pathway that produces fatty acid derivatives, for example,fatty alcohols. The fatty acid biosynthetic pathway includes fatty acidsynthases that can be engineered to produce fatty acids, and in someembodiments can be expressed with additional enzymes to produce fattyacid derivatives, such as fatty alcohols having desired characteristics.

As used herein, “fatty aldehyde” means an aldehyde having the formulaRCHO characterized by a carbonyl group (C═O). In some embodiments, thefatty aldehyde is any aldehyde made from a fatty alcohol. In certainembodiments, the R group is at least 5, at least 6, at least 7, at least8, at least 9, at least 10, at least 11, at least 12, at least 13, atleast 14, at least 15, at least 16, at least 17, at least 18, or atleast 19, carbons in length. Alternatively, or in addition, the R groupis 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 orless, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 orless, 8 or less, 7 or less, or 6 or less carbons in length. Thus, the Rgroup can have an R group hounded by any two of the above endpoints. Forexample, the R group can be 6-16 carbons in length, 10-14 carbons inlength, or 12-18 carbons in length. In some embodiments, the fattyaldehyde is a C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇,C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, C₂₄, C₂₅, or a C₂₆ fatty aldehyde. Incertain embodiments, the fatty aldehyde is a C₆, C₈, C₁₀, C₁₂, C₁₃, C₁₄,C₁₅, C₁₆, C₁₇, or C₁₈ fatty aldehyde.

As used herein, “fatty alcohol” means an alcohol having the formula ROH.In some embodiments, the R group is at least 5, at least 6, at least 7,at least 8, at least 9, at least 10, at least 11, at least 12, at least13, at least 14, at least 15, at least 16, at least 17, at least 18, orat least 19, carbons in length. Alternatively, or in addition, the Rgroup is 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9or less, 8 or less, 7 or less, or 6 or less carbons in length. Thus, theR group can have an R group bounded by any two of the above endpoints.For example, the R group can be 6-16 carbons in length, 10-14 carbons inlength, or 12-18 carbons in length. In some embodiments, the fattyalcohol is a C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇,C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, C₂₄, C₂₅, or a C₂₆ fatty alcohol. Incertain embodiments, the fatty alcohol is a C₆, C₈, C₁₀, C₁₂, C₁₃, C₁₄,C₁₅, C₁₆, C₁₇, or C₁₈ fatty alcohol.

A “fatty alcohol composition” as referred to herein is produced by arecombinant host cell and typically comprises a mixture of fattyalcohols. In some cases, the mixture includes more than one type ofproduct (e.g., fatty alcohols and fatty acids). In other cases, thefatty acid derivative compositions may comprise, for example, a mixtureof fatty alcohols with various chain lengths and saturation or branchingcharacteristics. In still other cases, the fatty alcohol compositioncomprises a mixture of both more than one type of product and productswith various chain lengths and saturation or branching characteristics.

A host cell engineered to produce a fatty aldehyde will typicallyconvert some of the fatty aldehyde to a fatty alcohol. When a host cell,which produces fatty alcohols is engineered to express a polynucleotideencoding an ester synthase, wax esters are produced. In one embodiment,fatty alcohols are made from a fatty acid biosynthetic pathway. As anexample, Acyl-ACP can be converted to fatty acids via the action of athioesterase (e.g., E. coli TesA), which are converted to fattyaldehydes and fatty alcohols via the action of a carboxylic acidreductase (e.g., E. coli CarB). Conversion of fatty aldehydes to fattyalcohols can be further facilitated, for example, via the action of afatty alcohol biosynthetic polypeptide. In some embodiments, a geneencoding a fatty alcohol biosynthetic polypeptide is expressed oroverexpressed in the host cell. In certain embodiments, the fattyalcohol biosynthetic polypeptide has aldehyde reductase or alcoholdehydrogenase activity. Examples of alcohol dehydrogenase polypeptidesuseful in accordance with the disclosure include, but are not limited toAlrA of Acinetobacter sp. M-1 (SEQ ID NO: 3) or AlrA homologs, such asAlrAadp1 (SEQ ID NO:4) and endogenous E. coli alcohol dehydrogenasessuch as YjgB, (AAC77226) (SEQ ID NO: 5), DkgA (NP_417485), DkgB(NP_414743), YdjL (AAC74846), YdjJ (NP_416288), AdhP (NP_415995), YhdH(NP_417719), YahK (NP_414859), YphC (AAC75598), YqhD (446856) and YbbO[AAC73595.1]. Additional examples are described in International PatentApplication Publication Nos. WO2007/136762, WO2008/119082 andWO2010/062480, each of which is expressly incorporated by referenceherein. In certain embodiments, the fatty alcohol biosyntheticpolypeptide has aldehyde reductase or alcohol dehydrogenase activity (EC1.1.1.1).

As used herein, the term “alcohol dehydrogenase” refers to a polypeptidecapable of catalyzing the conversion of a fatty aldehyde to an alcohol(e.g., fatty alcohol). One of ordinary skill in the art will appreciatethat certain alcohol dehydrogenases are capable of catalyzing otherreactions as well, and these non-specific alcohol dehydrogenases alsoare encompassed by the term “alcohol dehydrogenase.” The R group of afatty acid, fatty aldehyde, or fatty alcohol can be a straight chain ora branched chain. Branched chains may have more than one point ofbranching and may include cyclic branches. In some embodiments, thebranched fatty acid, branched fatty aldehyde, or branched fatty alcoholis a C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉,C₂₀, C₂₁, C₂₂, C₂₃, C₂₄, C₂₅, or a C₂₆ branched fatty acid, branchedfatty aldehyde, or branched fatty alcohol. In particular embodiments,the branched fatty acid, branched fatty aldehyde, or branched fattyalcohol is a C₆, C₈, C₁₀, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, or C₁₈ branchedfatty acid, branched fatty aldehyde, or branched fatty alcohol. Incertain embodiments, the hydroxyl group of the branched fatty acid,branched fatty aldehyde, or branched fatty alcohol is in the primary(C₁) position. In certain embodiments, the branched fatty acid, branchedfatty aldehyde, or branched fatty alcohol is an iso-fatty acid,iso-fatty aldehyde, or iso-fatty alcohol, or an anteiso-fatty acid, ananteiso-fatty aldehyde, or anteiso-fatty alcohol. In exemplaryembodiments, the branched fatty acid, branched fatty aldehyde, orbranched fatty alcohol is selected from iso-C_(7:0), iso-C_(8:0),iso-C_(9:0), iso-C_(10:0), iso-C_(12:0), iso-C_(13:0), iso-C_(14:0),iso-C_(15:0), iso-C_(16:0), iso-C_(17:0), iso-C_(18:0), iso-C_(19:0),anteiso-C_(7:0), anteiso-C_(8:0), anteiso-C_(9:0), anteiso-C_(10:0),anteiso-C_(11:0), anteiso-C_(12:0), anteiso-C_(13:0), anteiso-C_(14:0),anteiso-C_(15:0), anteiso-C_(16:0), anteiso-C_(17:0), anteiso-C_(18:0),and anteiso-C_(19:0) branched fatty acid, branched fatty aldehyde orbranched fatty alcohol. The R group of a branched or unbranched fattyacid, branched or unbranched fatty aldehyde, or branched or unbranchedfatty alcohol can be saturated or unsaturated. If unsaturated, the Rgroup can have one or more than one point of unsaturation. In someembodiments, the unsaturated fatty acid, unsaturated fatty aldehyde, orunsaturated fatty alcohol is a monounsaturated fatty acid,monounsaturated fatty aldehyde, or monounsaturated fatty alcohol. Incertain embodiments, the unsaturated fatty acid, unsaturated fattyaldehyde, or unsaturated fatty alcohol is a C6:1, C7:1, C8:1, C9:1,C10:1, C11:1, C12:1, C13:1, C14:1, C15:1, C16:1, C17:1, C18:1, C19:1,C20:1, C21:1, C22:1, C23:1, C24:1, C25:1, or a C26:1 unsaturated fattyacid, unsaturated fatty aldehyde, or unsaturated fatty alcohol. Incertain preferred embodiments, the unsaturated fatty acid, unsaturatedfatty aldehyde, or unsaturated fatty alcohol is C10:1, C12:1, C14:1,C16:1, or C18:1. In yet other embodiments, the unsaturated fatty acid,unsaturated fatty aldehyde, or unsaturated fatty alcohol is unsaturatedat the omega-7 position. In certain embodiments, the unsaturated fattyacid, unsaturated fatty aldehyde, or unsaturated fatty alcohol comprisesa cis double bond.

As used herein, a recombinant or engineered “host cell” is a host cell,e.g., a microorganism that has been modified such that it produces fattyalcohols. In some embodiments, the recombinant host cell comprises oneor more polynucleotides, each polynucleotide encoding a polypeptidehaving fatty aldehyde and/or fatty alcohol biosynthetic enzyme activity,wherein the recombinant host cell produces a fatty alcohol compositionwhen cultured in the presence of a carbon source under conditionseffective to express the polynucleotides.

As used herein, the term “clone” typically refers to a cell or group ofcells descended from and essentially genetically identical to a singlecommon ancestor, for example, the bacteria of a cloned bacterial colonyarose from a single bacterial cell.

As used herein, the term “culture” typical refers to a liquid mediacomprising viable cells. In one embodiment, a culture comprises cellsreproducing in a predetermined culture media under controlledconditions, for example, a culture of recombinant host cells grown inliquid media comprising a selected carbon source and nitrogen.“Culturing” or “cultivation” refers to growing a population of microbialcells under suitable conditions in a liquid or solid medium. Inparticular embodiments, culturing refers to the fermentativebioconversion of a substrate to an end-product. Culturing media are wellknown and individual components of such culture media are available fromcommercial sources, e.g., under the Difco™ and BBL™ trademarks. In onenon-limiting example, the aqueous nutrient medium is a “rich medium”comprising complex sources of nitrogen, salts, and carbon, such as YPmedium, comprising 10 g/L of peptone and 10 g/L yeast extract of such amedium. The host cell can be additionally engineered to assimilatecarbon efficiently and use cellulosic materials as carbon sourcesaccording to methods described for example in U.S. Pat. Nos. 5,000,000;5,028,539; 5,424,202; 5,482,846; 5,602,030 and WO2010127318, each ofwhich is expressly incorporated by reference herein. In addition, thehost cell can be engineered to express an invertase so that sucrose canbe used as a carbon source.

As used herein, the term “under conditions effective to express saidheterologous nucleotide sequences” means any conditions that allow ahost cell to produce a desired fatty aldehyde or fatty alcohol. Suitableconditions include, for example, fermentation conditions.

As used herein, “modified” or an “altered level of” activity of aprotein, for example an enzyme, in a recombinant host cell refers to adifference in one or more characteristics in the activity determinedrelative to the parent or native host cell. Typically differences inactivity are determined between a recombinant host cell, having modifiedactivity, and the corresponding wild-type host cell (e.g., comparison ofa culture of a recombinant host cell relative to wild-type host cell).Modified activities can be the result of, for example, modified amountsof protein expressed by a recombinant host cell (e.g., as the result ofincreased or decreased number of copies of DNA sequences encoding theprotein, increased or decreased number of mRNA transcripts encoding theprotein, and/or increased or decreased amounts of protein translation ofthe protein from mRNA); changes in the structure of the protein (e.g.,changes to the primary structure, such as, changes to the protein'scoding sequence that result in changes in substrate specificity, changesin observed kinetic parameters); and changes in protein stability (e.g.,increased or decreased degradation of the protein). In some embodiments,the polypeptide is a mutant or a variant of any of the polypeptidesdescribed herein. In certain instances, the coding sequences for thepolypeptides described herein are codon optimized for expression in aparticular host cell. For example, for expression in E. coli, one ormore codons can be optimized as described in, e.g., Grosjean et al.,Gene 18:199-209 (1982).

The term “regulatory sequences” as used herein typically refers to asequence of bases in DNA, operably-linked to DNA sequences encoding aprotein that ultimately controls the expression of the protein. Examplesof regulatory sequences include, but are not limited to, RNA promotersequences, transcription factor binding sequences, transcriptiontermination sequences, modulators of transcription (such as enhancerelements), nucleotide sequences that affect RNA stability, andtranslational regulatory sequences (such as, ribosome binding sites(e.g., Shine-Dalgarno sequences in prokaryotes or Kozak sequences ineukaryotes), initiation codons, termination codons).

As used herein, the phrase “the expression of said nucleotide sequenceis modified relative to the wild type nucleotide sequence,” means anincrease or decrease in the level of expression and/or activity of anendogenous nucleotide sequence or the expression and/or activity of aheterologous or non-native polypeptide-encoding nucleotide sequence. Asused herein, the term “overexpress” means to express or cause to beexpressed a polynucleotide or polypeptide in a cell at a greaterconcentration than is normally expressed in a corresponding wild-typecell under the same conditions.

The terms “altered level of expression” and “modified level ofexpression” are used interchangeably and mean that a polynucleotide,polypeptide, or hydrocarbon is present in a different concentration inan engineered host cell as compared to its concentration in acorresponding wild-type cell under the same conditions.

As used herein, the term “titer” refers to the quantity of fattyaldehyde or fatty alcohol produced per unit volume of host cell culture.In any aspect of the compositions and methods described herein, a fattyalcohol is produced at a titer of about 25 mg/L, about 50 mg/L, about 75mg/L, about 100 mg/L, about 125 mg/L, about 150 mg/L, about 175 mg/L,about 200 mg/L, about 225 mg/L, about 250 mg/L, about 275 mg/L, about300 mg/L, about 325 mg/L, about 350 mg/L, about 375 mg/L, about 400mg/L, about 425 mg/L, about 450 mg/L, about 475 mg/L, about 500 mg/L,about 525 mg/L, about 550 mg/L, about 575 mg/L, about 600 mg/L, about625 mg/L, about 650 mg/L, about 675 mg/L, about 700 mg/L, about 725mg/L, about 750 mg/L, about 775 mg/L, about 800 mg/L, about 825 mg/L,about 850 mg/L, about 875 mg/L, about 900 mg/L, about 925 mg/L, about950 mg/L, about 975 mg/L, about 1000 mg/L, about 1050 mg/L, about 1075mg/L, about 1100 mg/L, about 1125 mg/L, about 1150 mg/L, about 1175mg/L, about 1200 mg/L, about 1225 mg/L, about 1250 mg/L, about 1275mg/L, about 1300 mg/L, about 1325 mg/L, about 1350 mg/L, about 1375mg/L, about 1400 mg/L, about 1425 mg/L, about 1450 mg/L, about 1475mg/L, about 1500 mg/L, about 1525 mg/L, about 1550 mg/L, about 1575mg/L, about 1600 mg/L, about 1625 mg/L, about 1650 mg/L, about 1675mg/L, about 1700 mg/L, about 1725 mg/L, about 1750 mg/L, about 1775mg/L, about 1800 mg/L, about 1825 mg/L, about 1850 mg/L, about 1875mg/L, about 1900 mg/L, about 1925 mg/L, about 1950 mg/L, about 1975mg/L, about 2000 mg/L (2 g/L), 3 g/L, 5 g/L, 10 g/L, 20 g/L, 30 g/L, 40g/L, 50 g/L, 60 g/L, 70 g/L, 80 g/L, 90 g/L, 100 g/L or a range boundedby any two of the foregoing values. In other embodiments, a fattyaldehyde or fatty alcohol is produced at a titer of more than 100 g/L,more than 200 g/L, more than 300 g/L, or higher, such as 500 g/L, 700g/L, 1000 g/L, 1200 g/L, 1500 g/L, or 2000 g/L. The preferred titer offatty aldehyde or fatty alcohol produced by a recombinant host cellaccording to the methods of the disclosure is from 5 g/L to 200 g/L, 10g/L to 150 g/L, 20 g/L to 120 g/L and 30 g/L to 100 g/L.

As used herein, the term “yield of the fatty aldehyde or fatty alcoholproduced by a host cell” refers to the efficiency by which an inputcarbon source is converted to product (i.e., fatty alcohol or fattyaldehyde) in a host cell. Host cells engineered to produce fattyalcohols and/or fatty aldehydes according to the methods of thedisclosure have a yield of at least 3%, at least 4%, at least 5%, atleast 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least11%, at least 12%, at least 13%, at least 14%, at least 15%, at least16%, at least 17%, at least 18%, at least 19%, at least 20%, at least21%, at least 22%, at least 23%, at least 24%, at least 25%, at least26%, at least 27%, at least 28%, at least 29%, or at least 30% or arange bounded by any two of the foregoing values. In other embodiments,a fatty aldehyde or fatty alcohol is produced at a yield of more than30%, 40%, 50%, 60%, 70%, 80%, 90% or more. Alternatively, or inaddition, the yield is about 30% or less, about 27% or less, about 25%or less, or about 22% or less. Thus, the yield can be bounded by any twoof the above endpoints. For example, the yield of the fatty alcohol orfatty aldehyde produced by the recombinant host cell according to themethods of the disclosure can be 5% to 15%, 10% to 25%, 10% to 22%, 15%to 27%, 18% to 22%, 20% to 28%, or 20% to 30%. The preferred yield offatty alcohol produced by the recombinant host cell according to themethods of the disclosure is from 10% to 30%.

As used herein, the term “productivity” refers to the quantity of fattyaldehyde or fatty alcohol produced per unit volume of host cell cultureper unit time. In any aspect of the compositions and methods describedherein, the productivity of fatty aldehyde or fatty alcohol produced bya recombinant host cell is at least 100 mg/L/hour, at least 200mg/L/hour₀, at least 300 mg/L/hour, at least 400 mg/L/hour, at least 500mg/L/hour, at least 600 mg/L/hour, at least 700 mg/L/hour, at least 800mg/L/hour, at least 900 mg/L/hour, at least 1000 mg/L/hour, at least1100 mg/L/hour, at least 1200 mg/L/hour, at least 1300 mg/L/hour, atleast 1400 mg/L/hour, at least 1500 mg/L/hour, at least 1600 mg/L/hour,at least 1700 mg/L/hour, at least 1800 mg/L/hour, at least 1900mg/L/hour, at least 2000 mg/L/hour, at least 2100 mg/L/hour, at least2200 mg/L/hour, at least 2300 mg/L/hour, at least 2400 mg/L/hour, or atleast 2500 mg/L/hour. Alternatively, or in addition, the productivity is2500 mg/L/hour or less, 2000 mg/L/OD₆₀₀ or less, 1500 mg/L/OD₆₀₀ orless, 120 mg/L/hour, or less, 1000 mg/L/hour or less, 800 mg/L/hour, orless, or 600 mg/L/hour or less. Thus, the productivity can be bounded byany two of the above endpoints. For example, the productivity can be 3to 30 mg/L/hour₀, 6 to 20 mg/L/hour, or 15 to 30 mg/L/hour. Thepreferred productivity of a fatty aldehyde or fatty alcohol produced bya recombinant host cell according to the methods of the disclosure isselected from 500 mg/L/hour to 2500 mg/L/hour, or from 700 mg/L/hour to2000 mg/L/hour.

The terms “total fatty species” and “total fatty acid product” may beused interchangeably herein with reference to the total amount of fattyalcohols, fatty aldehydes, free fatty acids, and fatty esters present ina sample as evaluated by GC-FID as described in International PatentApplication Publication WO 2008/119082. Samples may contain one, two,three, or four of these compounds depending on the context.

As used herein, the term “glucose utilization rate” means the amount ofglucose used by the culture per unit time, reported as grams/liter/hour(g/L/hr).

As used herein, the term “carbon source” refers to a substrate orcompound suitable to be used as a source of carbon for prokaryotic orsimple eukaryotic cell growth. Carbon sources can be in various forms,including, but not limited to polymers, carbohydrates, acids, alcohols,aldehydes, ketones, amino acids, peptides, and gases (e.g., CO and CO₂).Exemplary carbon sources include, but are not limited to,monosaccharides, such as glucose, fructose, mannose, galactose, xylose,and arabinose; oligosaccharides, such as fructo-oligosaccharide andgalacto-oligosaccharide; polysaccharides such as starch, cellulose,pectin, and xylan; disaccharides, such as sucrose, maltose, cellobiose,and turanose; cellulosic material and variants such as hemicelluloses,methyl cellulose and sodium carboxymethyl cellulose; saturated orunsaturated fatty acids, succinate, lactate, and acetate; alcohols, suchas ethanol, methanol, and glycerol, or mixtures thereof. The carbonsource can also be a product of photosynthesis, such as glucose. Incertain preferred embodiments, the carbon source is biomass. In otherpreferred embodiments, the carbon source is glucose. In other preferredembodiments the carbon source is sucrose.

As used herein, the term “biomass” refers to any biological materialfrom which a carbon source is derived. In some embodiments, a biomass isprocessed into a carbon source, which is suitable for bioconversion. Inother embodiments, the biomass does not require further processing intoa carbon source. The carbon source can be converted into a biofuel. Anexemplary source of biomass is plant matter or vegetation, such as corn,sugar cane, or switchgrass. Another exemplary source of biomass ismetabolic waste products, such as animal matter (e.g., cow manure).Further exemplary sources of biomass include algae and other marineplants. Biomass also includes waste products from industry, agriculture,forestry, and households, including, but not limited to, fermentationwaste, ensilage, straw, lumber, sewage, garbage, cellulosic urban waste,and food leftovers. The term “biomass” also can refer to sources ofcarbon, such as carbohydrates (e.g., monosaccharides, disaccharides, orpolysaccharides).

As used herein, the term “isolated,” with respect to products (such asfatty acids and derivatives thereof) refers to products that areseparated from cellular components, cell culture media, or chemical orsynthetic precursors. The fatty acids and derivatives thereof producedby the methods described herein can be relatively immiscible in thefermentation broth, as well as in the cytoplasm. Therefore, the fattyacids and derivatives thereof can collect in an organic phase eitherintracellularly or extracellularly.

As used herein, the terms “purify,” “purified,” or “purification” meanthe removal or isolation of a molecule from its environment by, forexample, isolation or separation. “Substantially purified” molecules areat least about 60% free (e.g., at least about 70% free, at least about75% free, at least about 85% free, at least about 90% free, at leastabout 95% free, at least about 97% free, at least about 99% free) fromother components with which they are associated. As used herein, theseterms also refer to the removal of contaminants from a sample. Forexample, the removal of contaminants can result in an increase in thepercentage of a fatty aldehyde or a fatty alcohol in a sample. Forexample, when a fatty aldehyde or a fatty alcohol is produced in arecombinant host cell, the fatty aldehyde or fatty alcohol can bepurified by the removal of recombinant host cell proteins. Afterpurification, the percentage of a fatty aldehyde or a fatty alcohol inthe sample is increased. The terms “purify,” “purified,” and“purification” are relative terms which do not require absolute purity.Thus, for example, when a fatty aldehyde or a fatty alcohol is producedin recombinant host cells, a purified fatty aldehyde or a purified fattyalcohol is a fatty aldehyde or a fatty alcohol that is substantiallyseparated from other cellular components (e.g., nucleic acids,polypeptides, lipids, carbohydrates, or other hydrocarbons).

Strain Improvements

In order to meet very high targets for titer, yield, and/or productivityof fatty alcohols, a number of modifications were made to the productionhost cells. FadR is a key regulatory factor involved in fatty aciddegradation and fatty acid biosynthesis pathways (Cronan et al., Mol.Microbiol., 29(4): 937-943 (1998)). The E. coli ACS enzyme FadD and thefatty acid transport protein FadL are essential components of a fattyacid uptake system. FadL mediates transport of fatty acids into thebacterial cell, and FadD mediates formation of acyl-CoA esters. When noother carbon source is available, exogenous fatty acids are taken up bybacteria and converted to acyl-CoA esters, which can bind to thetranscription factor FadR and derepress the expression of the fad genesthat encode proteins responsible for fatty acid transport (FadL),activation (FadD), and β-oxidation (FadA, FadB, FadE, and FadH). Whenalternative sources of carbon are available, bacteria synthesize fattyacids as acyl-ACPs, which are used for phospholipid synthesis, but arenot substrates for β-oxidation. Thus, acyl-CoA and acyl-ACP are bothindependent sources of fatty acids that can result in differentend-products (Caviglia et al., J. Biol. Chem., 279(12): 1163-1169(2004)). U.S. Provisional Application No. 61/470,989 describes improvedmethods of producing fatty acid derivatives in a host cell which isgenetically engineered to have an altered level of expression of a FadRpolypeptide as compared to the level of expression of the FadRpolypeptide in a corresponding wild-type host cell.

There are conflicting speculations in the art as to the limiting factorsof fatty acid biosynthesis in host cells, such as E. coli. One approachto increasing the flux through fatty acid biosynthesis is to manipulatevarious enzymes in the pathway (FIGS. 1 and 2). The supply of acyl-ACPsfrom acetyl-CoA via the acetyl-CoA carboxylase (acc) complex (FIG. 3)and fatty acid biosynthetic (fab) pathway may limit the rate of fattyalcohol production. In one exemplary approach detailed in Example 2, theeffect of overexpression of Corynebacterium glutamicum accABCD (±birA)demonstrated that such genetic modifications can lead to increasedacetyl-coA and malonyl-CoA in E. coli. One possible reason for a lowrate of flux through fatty acid biosynthesis is a limited supply ofprecursors, namely acetyl-CoA and, in particular, malonyl-CoA, and themain precursors for fatty acid biosynthesis. Example 3 describes theconstruction of fab operons that encode enzymes in the biosyntheticpathway for conversion of malonyl-CoA into acyl-ACPs and integrationinto the chromosome of an E. coli host cell. In yet another approachdetailed in Example 4, mutations in the rph and ilvG genes in the E.coli host cell were shown to result in higher free fatty acid (FFA)production, which translated into higher production of fatty alcohol. Instill another approach, transposon mutagenesis and high-throughputscreening was done to find beneficial mutations that increase the titeror yield. Example 5 describes how a transposon insertion in the yijPgene can improve the fatty alcohol yield in shake flask and fed-batchfermentations.

Carboxylic Acid Reductase (CAR)

Recombinant host cells have been engineered to produce fatty alcohols byexpressing a thioesterase, which catalyzes the conversion of acyl-ACPsinto free fatty acids (FFAs) and a carboxylic acid reductase (CAR),which converts free fatty acids into fatty aldehydes. Native(endogenous) aldehyde reductases present in the host cell (e.g., E.coli) can convert fatty aldehydes into fatty alcohols. Exemplarythioesterases are described for example in US Patent Publication No.20100154293, expressly incorporated by reference herein. CarB, is anexemplary carboxylic acid reductase, a key enzyme in the fatty alcoholproduction pathway. WO2010/062480 describes a BLAST search using theNRRL 5646 CAR amino acid sequence (Genpept accession AAR91681) (SEQ IDNO: 6) as the query sequence, and use thereof in identification ofapproximately 20 homologous sequences.

The terms “carboxylic acid reductase,” “CAR,” and “fatty aldehydebiosynthetic polypeptide” are used interchangeably herein. In practicingthe disclosure, a gene encoding a carboxylic acid reductase polypeptideis expressed or overexpressed in the host cell. In some embodiments, theCarB polypeptide has the amino acid sequence of SEQ ID NO: 7. In otherembodiments, the CarB polypeptide is a variant or mutant of SEQ ID NO:7. In certain embodiments, the CarB polypeptide is from a mammaliancell, plant cell, insect cell, yeast cell, fungus cell, filamentousfungi cell, a bacterial cell, or any other organism. In someembodiments, the bacterial cell is a mycobacterium selected from thegroup consisting of Mycobacterium smegmatis, Mycobacterium abscessus,Mycobacterium avium, Mycobacterium bovis, Mycobacterium tuberculosis,Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium ulcerans.In other embodiments, the bacterial cell is from a Nocardia species, forexample, Nocardia NRRL 5646, Nocardia farcinica, Streptomyces griseus,Salinispora arenicola, or Clavibacter michiganenesis. In otherembodiments, the CarB polypeptide is a homologue of CarB having an aminoacid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to the amino acid sequence of SEQ ID NO: 7. The identity of aCarB polypeptide having at least about 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identity to the amino acid sequence of SEQ ID NO: 7 is not particularlylimited, and one of ordinary skill in the art can readily identifyhomologues of E. coli MG1655 derived-CarB and determine its functionusing the methods described herein. In other embodiments, the CarBpolypeptide contains a mutation at amino acid number 3, 12, 20, 28, 46,74, 103, 191, 288, 473, 827, 926, 927, 930 or 1128 of SEQ ID NO: 7.Exemplary mutations are detailed in Table 10. Preferred fragments ormutants of a polypeptide retain some or all of the biological function(e.g., enzymatic activity) of the corresponding wild-type polypeptide.In some embodiments, the fragment or mutant retains at least about 75%,at least about 80%, at least about 90%, at least about 95%, or at leastabout 98% or more of the biological function of the correspondingwild-type polypeptide. In other embodiments, the fragment or mutantretains about 100% of the biological function of the correspondingwild-type polypeptide. Guidance in determining which amino acid residuesmay be substituted, inserted, or deleted without affecting biologicalactivity may be found using computer programs well known in the art, forexample, LASERGENE™ software (DNASTAR, Inc., Madison, Wis.).

In yet other embodiments, a fragment or mutant exhibits increasedbiological function as compared to a corresponding wild-typepolypeptide. For example, a fragment or mutant may display at leastabout a 10%, at least about a 25%, at least about a 50%, at least abouta 75%, or at least about a 90% improvement in enzymatic activity ascompared to the corresponding wild-type polypeptide. In otherembodiments, the fragment or mutant displays at least about 100% (e.g.,at least about 200%, or at least about 500%) improvement in enzymaticactivity as compared to the corresponding wild-type polypeptide. It isunderstood that the polypeptides described herein may have additionalconservative or non-essential amino acid substitutions, which do nothave a substantial effect on the polypeptide function. Whether or not aparticular substitution will be tolerated (i.e., will not adverselyaffect desired biological function, such as DNA binding or enzymeactivity) can be determined as described in Bowie et al. (Science, 247:1306-1310 (1990)).

As a result of the methods and variant enzymes of the presentdisclosure, one or more of the titer, yield, and/or productivity of thefatty acid or derivative thereof produced by the engineered host cellhaving an altered level of expression of a CarB polypeptide is increasedrelative to that of the corresponding wild-type host cell. To allow formaximum conversion of C12 and C14 fatty acids into fatty alcohols, CarBmust be expressed at sufficient activity. An improved recombinant hostcell would have a CAR enzyme that is expressed from, for example, the E.coli chromosome. As shown in Example 6, cells expressing the CarB enzymefrom the chromosome have more carboxylic acid reductase activityrelative to the original CarB and are able to convert more C12 and C14fatty acids into fatty alcohols. CarB is a large gene (3.5 kb) andincreases plasmid size considerably, making it difficult to use a pCLplasmid to test new genes during strain development. Approaches toincreasing the activity of CarB, include increasing its solubility,stability, expression and/or functionality. In one exemplary approach, afusion protein that contains 6 histidines and a thrombin cleavage siteat the N-terminus of CarB is produced. This enzyme differs from CarB byan additional 60 nucleotides at the N-terminus, and is named CarB60.When CarB or CarB60 are expressed from the E. coli chromosome undercontrol of the pTRC promoter, cells containing CarB60 have increasedtotal cellular carboxylic acid reductase activity and convert more C12and C14 free fatty acids (FFAs) into fatty alcohols. One of skill in theart will appreciate that this is one example of molecular engineering inorder to achieve a greater conversion of C12 and C14 free fatty acids(FFAs) into fatty alcohols as illustrated in Example 6 (supra). Similarapproaches are encompassed herein (see Example 7).

Phosphopantetheine transferases (PPTases) (EC 2.7.8.7) catalyze thetransfer of 4′-phosphopantetheine from CoA to a substrate. Nocardia Car,CarB and several homologues thereof contain a putative attachment sitefor 4% phosphopantetheine (PPT) (He et al., Appl. Environ. Microbial.,70(3): 1874-1881 (2004)). In some embodiments of the disclosure, aPPTase is expressed or overexpressed in an engineered host cell. Incertain embodiments, the PPTase is EntD from E. coli MG1655 (SEQ IDNO:8). In some embodiments, a thioesterase and a carboxylic acidreductase are expressed or overexpressed in an engineered host cell. Incertain embodiments, the thioesterase is tesA and the carboxylic acidreductase is carB. In other embodiments, a thioesterase, a carboxylicacid reductase and an alcohol dehydrogenase are expressed oroverexpressed in an engineered host cell. In certain embodiments, thethioesterase is tesA, the carboxylic acid reductase is carB and thealcohol dehydrogenase is alrAadp1 (GenPept accession number CAG70248.1)from Acinetobacter baylyi ADP1 (SEQ ID NO: 4). In still otherembodiments, a thioesterase, a carboxylic acid reductase, a PPTase, andan alcohol dehydrogenase are expressed or overexpressed in theengineered host cell. In certain embodiments, the thioesterase is tesA,the carboxylic acid reductase is carB, the PPTase is entD, and thealcohol dehydrogenase is alrAadp1. In still further embodiments, amodified host cell which expresses one or more of a thioesterase, a CAR,a PPTase, and an alcohol dehydrogenase also has one or more strainimprovements. Exemplary strain improvements include, but are not limitedto expression or overexpression of an acetyl-CoA carboxylasepolypeptide, overexpression of a FadR polypeptide, expression oroverexpression of a heterologous iFAB operon, or transposon insertion inthe yijP gene or another gene, or similar approaches. The disclosurealso provides a fatty alcohol composition produced by any of the methodsdescribed herein. A fatty alcohol composition produced by any of themethods described herein can be used directly as a starting materialsfor production of other chemical compounds (e.g., polymers, surfactants,plastics, textiles, solvents, adhesives, etc.), or personal careadditives. These compounds can also be used as feedstock for subsequentreactions, for example, hydrogenation, catalytic cracking (e.g., viahydrogenation, pyrolisis, or both) to make other products.

Mutants or Variants

In some embodiments, the polypeptide expressed in a recombinant hostcell is a mutant or a variant of any of the polypeptides describedherein. The terms “mutant” and “variant” as used herein refer to apolypeptide having an amino acid sequence that differs from a wild-typepolypeptide by at least one amino acid. For example, the mutant cancomprise one or more of the following conservative amino acidsubstitutions: replacement of an aliphatic amino acid, such as alanine,valine, leucine, and isoleucine, with another aliphatic amino acid;replacement of a serine with a threonine; replacement of a threoninewith a serine; replacement of an acidic residue, such as aspartic acidand glutamic acid, with another acidic residue; replacement of a residuebearing an amide group, such as asparagine and glutamine, with anotherresidue bearing an amide group; exchange of a basic residue, such aslysine and arginine, with another basic residue; and replacement of anaromatic residue, such as phenylalanine and tyrosine, with anotheraromatic residue. In some embodiments, the mutant polypeptide has about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100,or more amino acid substitutions, additions, insertions, or deletions.Preferred fragments or mutants of a polypeptide retain some or all ofthe biological function (e.g., enzymatic activity) of the correspondingwild-type polypeptide. In some embodiments, the fragment or mutantretains at least about 75%, at least about 80%, at least about 90%, atleast about 95%, or at least about 98% or more of the biologicalfunction of the corresponding wild-type polypeptide. In otherembodiments, the fragment or mutant retains about 100% of the biologicalfunction of the corresponding wild-type polypeptide. Guidance indetermining which amino acid residues may be substituted, inserted, ordeleted without affecting biological activity may be found usingcomputer programs well known in the art, for example, LASERGENE™software (DNASTAR, Inc., Madison, Wis.).

In yet other embodiments, a fragment or mutant exhibits increasedbiological function as compared to a corresponding wild-typepolypeptide. For example, a fragment or mutant may display at least a10%, at least a 25%, at least a 50%, at least a 75%, or at least a 90%improvement in enzymatic activity as compared to the correspondingwild-type polypeptide. In other embodiments, the fragment or mutantdisplays at least 100% (e.g., at least 200%, or at least 500%)improvement in enzymatic activity as compared to the correspondingwild-type polypeptide. It is understood that the polypeptides describedherein may have additional conservative or non-essential amino acidsubstitutions, which do not have a substantial effect on the polypeptidefunction. Whether or not a particular substitution will be tolerated(i.e., will not adversely affect desired biological function, such ascarboxylic acid reductase activity) can be determined as described inBowie et al. (Science, 247: 1306-1310 (1990)). A conservative amino acidsubstitution is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine), and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Variants can be naturallyoccurring or created in vitro. In particular, such variants can becreated using genetic engineering techniques, such as site directedmutagenesis, random chemical mutagenesis, Exonuclease III deletionprocedures, or standard cloning techniques. Alternatively, suchvariants, fragments, analogs, or derivatives can be created usingchemical synthesis or modification procedures.

Methods of making variants are well known in the art. These includeprocedures in which nucleic acid sequences obtained from naturalisolates are modified to generate nucleic acids that encode polypeptideshaving characteristics that enhance their value in industrial orlaboratory applications. In such procedures, a large number of variantsequences having one or more nucleotide differences with respect to thesequence obtained from the natural isolate are generated andcharacterized. Typically, these nucleotide differences result in aminoacid changes with respect to the polypeptides encoded by the nucleicacids from the natural isolates. For example, variants can be preparedby using random and site-directed mutagenesis. Random and site-directedmutagenesis are described in, for example, Arnold, Curr. Opin. Biotech.,4: 450-455 (1993). Random mutagenesis can be achieved using error pronePCR (see, e.g., Leung et al., Technique, 1: 11-15 (1989); and Caldwellet al., PCR Methods Applic., 2: 28-33 (1992)). In error prone PCR, PCRis performed under conditions where the copying fidelity of the DNApolymerase is low, such that a high rate of point mutations is obtainedalong the entire length of the PCR product. Briefly, in such procedures,nucleic acids to be mutagenized (e.g., a polynucleotide sequenceencoding a carboxylic reductase enzyme) are mixed with PCR primers,reaction buffer, MgCl₂, MnCl₂, Taq polymerase, and an appropriateconcentration of dNTPs for achieving a high rate of point mutation alongthe entire length of the PCR product. For example, the reaction can beperformed using 20 fmoles of nucleic acid to be mutagenized, 30 pmole ofeach PCR primer, a reaction buffer comprising 50 mM KCl, 10 mM Tris HCl(pH 8.3), 0.01% gelatin, 7 mM MgCl₂, 0.5 mM MnCl₂, 5 units of Taqpolymerase, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP, and 1 mM dTTP. PCR canbe performed for 30 cycles of 94° C. for 1 min, 45° C. for 1 min, and72° C. for 1 min. However, it will be appreciated that these parameterscan be varied as appropriate. The mutagenized nucleic acids are thencloned into an appropriate vector, and the activities of thepolypeptides encoded by the mutagenized nucleic acids are evaluated (seeExample 7). Site-directed mutagenesis can be achieved usingoligonucleotide-directed mutagenesis to generate site-specific mutationsin any cloned DNA of interest. Oligonucleotide mutagenesis is describedin, for example, Reidhaar-Olson et al., Science, 241: 53-57 (1988).Briefly, in such procedures a plurality of double strandedoligonucleotides bearing one or more mutations to be introduced into thecloned DNA are synthesized and inserted into the cloned DNA to bemutagenized (e.g., a polynucleotide sequence encoding a CARpolypeptide). Clones containing the mutagenized DNA are recovered, andthe activities of the polypeptides they encode are assessed. Anothermethod for generating variants is assembly PCR. Assembly PCR involvesthe assembly of a PCR product from a mixture of small DNA fragments. Alarge number of different PCR reactions occur in parallel in the samevial, with the products of one reaction priming the products of anotherreaction. Assembly PCR is described in, for example, U.S. Pat. No.5,965,408. Still another method of generating variants is sexual PCRmutagenesis. In sexual PCR mutagenesis, forced homologous recombinationoccurs between DNA molecules of different, but highly related, DNAsequences in vitro as a result of random fragmentation of the DNAmolecule based on sequence homology. This is followed by fixation of thecrossover by primer extension in a PCR reaction. Sexual PCR mutagenesisis described in, for example, Stemmer, Proc. Natl. Acad. Sci., USA., 91:10747-10751 (1994).

Variants can also be created by in vivo mutagenesis. In someembodiments, random mutations in a nucleic acid sequence are generatedby propagating the sequence in a bacterial strain, such as an E. colistrain, which carries mutations in one or more of the DNA repairpathways. Such “mutator” strains have a higher random mutation rate thanthat of a wild-type strain. Propagating a DNA sequence (e.g., apolynucleotide sequence encoding a CAR polypeptide) in one of thesestrains will eventually generate random mutations within the DNA.Mutator strains suitable for use for in vivo mutagenesis are describedin, for example, International Patent Application Publication No.WO1991/016427. Variants can also be generated using cassettemutagenesis. In cassette mutagenesis, a small region of adouble-stranded DNA molecule is replaced with a syntheticoligonucleotide “cassette” that differs from the native sequence. Theoligonucleotide often contains a completely and/or partially randomizednative sequence. Recursive ensemble mutagenesis can also be used togenerate variants. Recursive ensemble mutagenesis is an algorithm forprotein engineering (i.e., protein mutagenesis) developed to producediverse populations of phenotypically related mutants whose membersdiffer in amino acid sequence. This method uses a feedback mechanism tocontrol successive rounds of combinatorial cassette mutagenesis.Recursive ensemble mutagenesis is described in, for example, Arkin etal., Proc. Natl. Acad. Sci., U.S.A., 89: 7811-7815 (1992). In someembodiments, variants are created using exponential ensemblemutagenesis. Exponential ensemble mutagenesis is a process forgenerating combinatorial libraries with a high percentage of unique andfunctional mutants, wherein small groups of residues are randomized inparallel to identify, at each altered position, amino acids which leadto functional proteins. Exponential ensemble mutagenesis is describedin, for example, Delegrave et al., Biotech. Res, 11: 1548-1552 (1993).In some embodiments, variants are created using shuffling procedureswherein portions of a plurality of nucleic acids that encode distinctpolypeptides are fused together to create chimeric nucleic acidsequences that encode chimeric polypeptides as described in, forexample, U.S. Pat. Nos. 5,965,408 and 5,939,250.

Insertional mutagenesis is mutagenesis of DNA by the insertion of one ormore bases. Insertional mutations can occur naturally, mediated by virusor transposon, or can be artificially created for research purposes inthe lab, e.g., by transposon mutagenesis. When exogenous DNA isintegrated into that of the host, the severity of any ensuing mutationdepends entirely on the location within the host's genome wherein theDNA is inserted. For example, significant effects may be evident if atransposon inserts in the middle of an essential gene, in a promoterregion, or into a repressor or an enhancer region. Transposonmutagenesis and high-throughput screening was done to find beneficialmutations that increase the titer or yield of fatty alcohol. Thedisclosure provides recombinant host cells comprising (a) apolynucleotide sequence encoding a carboxylic acid reductase comprisingan amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to the amino acid sequence of SEQ ID NO: 7 and (b) apolynucleotide encoding a polypeptide having carboxylic acid reductaseactivity, wherein the recombinant host cell is capable of producing afatty aldehyde or a fatty alcohol.

Engineering Host Cells

In some embodiments, a polynucleotide (or gene) sequence is provided toa host cell by way of a recombinant vector, which comprises a promoteroperably linked to the polynucleotide sequence. In certain embodiments,the promoter is a developmentally-regulated, an organelle-specific, atissue-specific, an inducible, a constitutive, or a cell-specificpromoter. In some embodiments, the recombinant vector includes (a) anexpression control sequence operatively coupled to the polynucleotidesequence; (b) a selection marker operatively coupled to thepolynucleotide sequence; (c) a marker sequence operatively coupled tothe polynucleotide sequence; (d) a purification moiety operativelycoupled to the polynucleotide sequence; (e) a secretion sequenceoperatively coupled to the polynucleotide sequence; and (f) a targetingsequence operatively coupled to the polynucleotide sequence. Theexpression vectors described herein include a polynucleotide sequencedescribed herein in a form suitable for expression of the polynucleotidesequence in a host cell. It will be appreciated by those skilled in theart that the design of the expression vector can depend on such factorsas the choice of the host cell to be transformed, the level ofexpression of polypeptide desired, etc. The expression vectors describedherein can be introduced into host cells to produce polypeptides,including fusion polypeptides, encoded by the polynucleotide sequencesdescribed herein. Expression of genes encoding polypeptides inprokaryotes, for example, E. coli, is most often carried out withvectors containing constitutive or inducible promoters directing theexpression of either fusion or non-fusion polypeptides. Fusion vectorsadd a number of amino acids to a polypeptide encoded therein, usually tothe amino- or carboxy-terminus of the recombinant polypeptide. Suchfusion vectors typically serve one or more of the following threepurposes: (1) to increase expression of the recombinant polypeptide; (2)to increase the solubility of the recombinant polypeptide; and (3) toaid in the purification of the recombinant polypeptide by acting as aligand in affinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant polypeptide. This enables separation of therecombinant polypeptide from the fusion moiety after purification of thefusion polypeptide. Examples of such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin, and enterokinase.Exemplary fusion expression vectors include pGEX (Pharmacia Biotech,Inc., Piscataway, N.J.; Smith et al., Gene, 67: 31-40 (1988)), pMAL (NewEngland Biolabs, Beverly, Mass.), and pRITS (Pharmacia Biotech, Inc.,Piscataway, N.J.), which fuse glutathione S-transferase (GST), maltose Ebinding protein, or protein A, respectively, to the target recombinantpolypeptide.

Examples of inducible, non-fusion E. coli expression vectors includepTrc (Amann et al., Gene (1988) 69:301-315) and pET 11d (Studier et al.,Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990) 60-89). Target gene expression from the pTrcvector relies on host RNA polymerase transcription from a hybrid trp-lacfusion promoter. Target gene expression from the pET 11d vector relicson transcription from a T7 gn10-lac fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a resident λprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV 5 promoter. Suitable expression systems for both prokaryoticand eukaryotic cells are well known in the art; see, e.g., Sambrook etal., “Molecular Cloning: A Laboratory Manual,” second edition, ColdSpring Harbor Laboratory, (1989). Examples of inducible, non-fusion E.coli expression vectors include pTrc (Amann et al., Gene, 69: 301-315(1988)) and PET 11d (Studier et al., Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif., pp. 60-89 (1990)).In certain embodiments, a polynucleotide sequence of the disclosure isoperably linked to a promoter derived from bacteriophage T5. In oneembodiment, the host cell is a yeast cell. In this embodiment, theexpression vector is a yeast expression vector. Vectors can beintroduced into prokaryotic or eukaryotic cells via a variety ofart-recognized techniques for introducing foreign nucleic acid (e.g.,DNA) into a host cell. Suitable methods for transforming or transfectinghost cells can be found in, for example, Sambrook et al. (supra). Forstable transformation of bacterial cells, it is known that, dependingupon the expression vector and transformation technique used, only asmall fraction of cells will take-up and replicate the expressionvector. In some embodiments, in order to identify and select thesetransformants, a gene that encodes a selectable marker (e.g., resistanceto an antibiotic) is introduced into the host cells along with the geneof interest. Selectable markers include those that confer resistance todrugs such as, but not limited to, ampicillin, kanamycin,chloramphenicol, or tetracycline. Nucleic acids encoding a selectablemarker can be introduced into a host cell on the same vector as thatencoding a polypeptide described herein or can be introduced on aseparate vector. Cells stably transformed with the introduced nucleicacid can be identified by growth in the presence of an appropriateselection drug.

Production of Fatty Alcohol Compositions by Recombinant Host Cells

Strategies to increase production of fatty alcohols by recombinant hostcells include increased flux through the fatty acid biosynthetic pathwayby overexpression of native fatty acid biosynthesis genes and expressionof exogenous fatty acid biosynthesis genes from different organisms inan engineered production host. Enhanced activity of relevant enzymes inthe fatty alcohol biosynthetic pathway, e.g., CAR, as well as otherstrategies to optimize the growth and productivity of the host cell mayalso be employed to maximize production. In some embodiments, therecombinant host cell comprises a polynucleotide encoding a polypeptide(an enzyme) having fatty alcohol biosynthetic activity (i.e., a fattyalcohol biosynthetic polypeptide or a fatty alcohol biosyntheticenzyme), and a fatty alcohol is produced by the recombinant host cell. Acomposition comprising fatty alcohols (a fatty alcohol composition) maybe produced by culturing the recombinant host cell in the presence of acarbon source under conditions effective to express a fatty alcoholbiosynthetic enzyme. In some embodiments, the fatty alcohol compositioncomprises fatty alcohols, however, a fatty alcohol composition maycomprise other fatty acid derivatives. Typically, the fatty alcoholcomposition is recovered from the extracellular environment of therecombinant host cell, i.e., the cell culture medium. In one approach,recombinant host cells have been engineered to produce fatty alcohols byexpressing a thioesterase, which catalyzes the conversion of acyl-ACPsinto free fatty acids (FFAs) and a carboxylic acid reductase (CAR),which converts free fatty acids into fatty aldehydes. Native(endogenous) aldehyde reductases present in the host cell (e.g., E.coli) can convert the fatty aldehydes into fatty alcohols. In someembodiments, the fatty alcohol is produced by expressing oroverexpressing in the recombinant host cell a polynucleotide encoding apolypeptide having fatty alcohol biosynthetic activity which converts afatty aldehyde to a fatty alcohol. For example, an alcohol dehydrogenase(also referred to herein as an aldehyde reductase, e.g., EC 1.1.1.1),may be used in practicing the disclosure. As used herein, the term“alcohol dehydrogenase” refers to a polypeptide capable of catalyzingthe conversion of a fatty aldehyde to an alcohol (e.g., a fattyalcohol). One of ordinary skill in the art will appreciate that certainalcohol dehydrogenases are capable of catalyzing other reactions aswell, and these non-specific alcohol dehydrogenases also are encompassedby the term “alcohol dehydrogenase.” Examples of alcohol dehydrogenasepolypeptides useful in accordance with the disclosure include, but arenot limited to AlrAadp1 (SEQ ID NO: 4) or AlrA homologs and endogenousE. coli alcohol dehydrogenases such as YjgB, (AAC77226) (SEQ ID NO: 5),DkgA (NP_417485), DkgB (NP_414743), YdjL (AAC74846), YdjJ (NP_416288),AdhP (NP_415995), YhdH (NP_417719), YahK (NP_414859), YphC (AAC75598),YqhD (446856) and YbbO [AAC73595.1]. Additional examples are describedin International Patent Application Publication Nos. WO2007/136762,WO2008/119082 and WO 2010/062480, each of which is expresslyincorporated by reference herein. In certain embodiments, the fattyalcohol biosynthetic polypeptide has aldehyde reductase or alcoholdehydrogenase activity (EC 1.1.1.1). In another approach, recombinanthost cells have been engineered to produce fatty alcohols by expressingfatty alcohol forming acyl-CoA reductases or fatty acyl reductases(FARs) which convert fatty acyl-thioester substrates (e.g., fattyacyl-CoA or fatty acyl-ACP) to fatty alcohols. In some embodiments, thefatty alcohol is produced by expressing or overexpressing apolynucleotide encoding a polypeptide having fatty alcohol formingacyl-CoA reductase (FAR) activity in a recombinant host cell. Examplesof FAR polypeptides useful in accordance with this embodiment aredescribed in PCT Publication No. WO2010/062480, which is expresslyincorporated by reference herein.

Fatty alcohol may be produced via an acyl-CoA dependent pathwayutilizing fatty acyl-ACP and fatty acyl-CoA intermediates and anacyl-CoA independent pathway utilizing fatty acyl-ACP intermediates butnot a fatty acyl-CoA intermediate. In particular embodiments, the enzymeencoded by the over expressed gene is selected from a fatty acidsynthase, an acyl-ACP thioesterase, a fatty acyl-CoA synthase and anacetyl-CoA carboxylase. In some embodiments, the protein encoded by theover expressed gene is endogenous to the host cell. In otherembodiments, the protein encoded by the overexpressed gene isheterologous to the host cell. Fatty alcohols are also made in nature byenzymes that are able to reduce various acyl-ACP or acyl-CoA moleculesto the corresponding primary alcohols. Sec also, U.S. Patent PublicationNos. 20100105963, and 20110206630 and U.S. Pat. No. 8,097,439, expresslyincorporated by reference herein. As used herein, a recombinant hostcell or an engineered host cell refers to a host cell whose geneticmakeup has been altered relative to the corresponding wild-type hostcell, for example, by deliberate introduction of new genetic elementsand/or deliberate modification of genetic elements naturally present inthe host cell. The offspring of such recombinant host cells also containthese new and/or modified genetic elements. In any of the aspects of thedisclosure described herein, the host cell can be selected from thegroup consisting of a plant cell, insect cell, fungus cell (e.g., afilamentous fungus, such as Candida sp., or a budding yeast, such asSaccharomyces sp.), an algal cell and a bacterial cell. In one preferredembodiment, recombinant host cells are recombinant microbial cells.Examples of host cells that are microbial cells, include but are notlimited to cells from the genus Escherichia, Bacillus, Lactobacillus,Zymomonas, Rhodococcus, Pseudomonas, Aspergillus, Trichoderma,Neurospora, Fusarium, Humicola, Rhizomucor, Kluyveromyces, Pichia,Mucor, Myceliophtora, Penicillium, Phanerochaete, Pleurotus, Trametes,Chrysosporium, Saccharomyces, Stenotrophamonas, Schizosaccharomyces,Yarrowia, or Streptomyces. In some embodiments, the host cell is aGram-positive bacterial cell. In other embodiments, the host cell is aGram-negative bacterial cell. In some embodiments, the host cell is anE. coli cell. In other embodiments, the host cell is a Bacillus lentuscell, a Bacillus brevis cell, a Bacillus stearothermophilus cell, aBacillus lichenoformis cell, a Bacillus alkalophilus cell, a Bacilluscoagulans cell, a Bacillus circulans cell, a Bacillus pumilis cell, aBacillus thuringiensis cell, a Bacillus clausii cell, a Bacillusmegaterium cell, a Bacillus subtilis cell, or a Bacillusamyloliquefaciens cell. In other embodiments, the host cell is aTrichoderma koningii cell, a Trichoderma viride cell, a Trichodermareesei cell, a Trichoderma longibrachiatum cell, an Aspergillus awamoricell, an Aspergillus fumigates cell, an Aspergillus foetidus cell, anAspergillus nidulans cell, an Aspergillus niger cell, an Aspergillusoryzae cell, a Humicola insolens cell, a Humicola lanuginose cell, aRhodococcus opacus cell, a Rhizomucor miehei cell, or a Mucor micheicell.

In yet other embodiments, the host cell is a Streptomyces lividans cellor a Streptomyces murinus cell. In yet other embodiments, the host cellis an Actinomycetes cell. In some embodiments, the host cell is aSaccharomyces cerevisiae cell. In some embodiments, the host cell is aSaccharomyces cerevisiae cell. In other embodiments, the host cell is acell from a eukaryotic plant, algae, cyanobacterium, green-sulfurbacterium, green non-sulfur bacterium, purple sulfur bacterium, purplenon-sulfur bacterium, extremophile, yeast, fungus, an engineeredorganism thereof, or a synthetic organism. In some embodiments, the hostcell is light-dependent or fixes carbon. In some embodiments, the hostcell is light-dependent or fixes carbon. In some embodiments, the hostcell has autotrophic activity. In some embodiments, the host cell hasphotoautotrophic activity, such as in the presence of light. In someembodiments, the host cell is heterotrophic or mixotrophic in theabsence of light. In certain embodiments, the host cell is a cell fromAvabidopsis thaliana, Panicum virgatum, Miscanthus giganteus, Zea mays,Botryococcuse braunii, Chlamydomonas reinhardtii, Dunaliela salina,Synechococcus Sp. PCC 7002, Synechococcus Sp. PCC 7942, SynechocystisSp. PCC 6803, Thermosynechococcus elongates BP-1, Chlorobium tepidum,Chlorojlexus auranticus, Chromatiumm vinosum, Rhodospirillum rubrum,Rhodobacter capsulatus, Rhodopseudomonas palusris, Clostridiumljungdahlii, Clostridiuthermocellum, Penicillium chrysogenum, Pichiapastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe,Pseudomonasjluorescens, or Zymomonas mobilis.

Culture and Fermentation of Engineered Host Cells

As used herein, fermentation broadly refers to the conversion of organicmaterials into target substances by host cells, for example, theconversion of a carbon source by recombinant host cells into fatty acidsor derivatives thereof by propagating a culture of the recombinant hostcells in a media comprising the carbon source. As used herein,conditions permissive for the production means any conditions that allowa host cell to produce a desired product, such as a fatty acid or afatty acid derivative. Similarly, conditions in which the polynucleotidesequence of a vector is expressed means any conditions that allow a hostcell to synthesize a polypeptide. Suitable conditions include, forexample, fermentation conditions. Fermentation conditions can comprisemany parameters, including but not limited to temperature ranges, levelsof aeration, feed rates and media composition. Each of these conditions,individually and in combination, allows the host cell to grow.Fermentation can be aerobic, anaerobic, or variations thereof (such asmicro-aerobic). Exemplary culture media include broths or gels.Generally, the medium includes a carbon source that can be metabolizedby a host cell directly. In addition, enzymes can be used in the mediumto facilitate the mobilization (e.g., the depolymerization of starch orcellulose to fermentable sugars) and subsequent metabolism of the carbonsource. For small scale production, the engineered host cells can begrown in batches of, for example, about 100 mL, 500 mL, 1 L, 2 L, 5 L,or 10 L; fermented; and induced to express a desired polynucleotidesequence, such as a polynucleotide sequence encoding a CAR polypeptide.For large scale production, the engineered host cells can be grown inbatches of about 10 L, 100 L, 1000 L, 10,000 L, 100,000 L, 1,000,000 Lor larger; fermented; and induced to express a desired polynucleotidesequence. Alternatively, large scale fed-batch fermentation may becarried out.

Fatty Alcohol Compositions

The fatty alcohol compositions described herein are found in theextracellular environment of the recombinant host cell culture and canbe readily isolated from the culture medium. A fatty alcohol compositionmay be secreted by the recombinant host cell, transported into theextracellular environment or passively transferred into theextracellular environment of the recombinant host cell culture. Thefatty alcohol composition is isolated from a recombinant host cellculture using routine methods known in the art. The disclosure providescompositions produced by engineered or recombinant host cells(bioproducts) which include one or more fatty aldehydes and/or fattyalcohols. Although a fatty alcohol component with a particular chainlength and degree of saturation may constitute the majority of thebioproduct produced by a cultured engineered or recombinant host cell,the composition typically includes a mixture of fatty aldehydes and/orfatty alcohols that vary with respect to chain length and/or degree ofsaturation. As used herein, fraction of modern carbon or f_(M) has thesame meaning as defined by National Institute of Standards andTechnology (NIST) Standard Reference Materials (SRMs 4990B and 4990C,known as oxalic acids standards HOxI and HOxII, respectively. Thefundamental definition relates to 0.95 times the ¹⁴C/¹²C isotope ratioHOxI (referenced to AD 1950). This is roughly equivalent todecay-corrected pre-Industrial Revolution wood. For the current livingbiosphere (plant material), f_(M) is approximately 1.1.

Bioproducts (e.g., the fatty aldehydes and alcohols produced inaccordance with the present disclosure) comprising biologically producedorganic compounds, and in particular, the fatty aldehydes and alcoholsbiologically produced using the fatty acid biosynthetic pathway herein,have not been produced from renewable sources and, as such, are newcompositions of matter. These new bioproducts can be distinguished fromorganic compounds derived from petrochemical carbon on the basis of dualcarbon-isotopic fingerprinting or ¹⁴C dating. Additionally, the specificsource of biosourced carbon (e.g., glucose vs. glycerol) can bedetermined by dual carbon-isotopic fingerprinting (see, e.g., U.S. Pat.No. 7,169,588, which is herein incorporated by reference). The abilityto distinguish bioproducts from petroleum based organic compounds isbeneficial in tracking these materials in commerce. For example, organiccompounds or chemicals comprising both biologically based and petroleumbased carbon isotope profiles may be distinguished from organiccompounds and chemicals made only of petroleum based materials. Hence,the bioproducts herein can be followed or tracked in commerce on thebasis of their unique carbon isotope profile. Bioproducts can bedistinguished from petroleum based organic compounds by comparing thestable carbon isotope ratio (¹³C/¹²C) in each fuel. The ¹³C/¹²C ratio ina given bioproduct is a consequence of the ¹³C/¹²C ratio in atmosphericcarbon dioxide at the time the carbon dioxide is fixed. It also reflectsthe precise metabolic pathway. Regional variations also occur.Petroleum, C₃ plants (the broadleaf), C₄ plants (the grasses), andmarine carbonates all show significant differences in ¹³C/¹²C and thecorresponding δ¹³C values. Furthermore, lipid matter of C₃ and C₄ plantsanalyze differently than materials derived from the carbohydratecomponents of the same plants as a consequence of the metabolic pathway.Within the precision of measurement, ¹³C shows large variations due toisotopic fractionation effects, the most significant of which forbioproducts is the photosynthetic mechanism. The major cause ofdifferences in the carbon isotope ratio in plants is closely associatedwith differences in the pathway of photosynthetic carbon metabolism inthe plants, particularly the reaction occurring during the primarycarboxylation (i.e., the initial fixation of atmospheric CO₂). Two largeclasses of vegetation are those that incorporate the C₃ (orCalvin-Benson) photosynthetic cycle and those that incorporate the C₄(or Hatch-Slack) photosynthetic cycle. In C₃ plants, the primary CO₂fixation or carboxylation reaction involves the enzymeribulose-1,5-diphosphate carboxylase, and the first stable product is a3-carbon compound. C₃ plants, such as hardwoods and conifers, aredominant in the temperate climate zones. In C₄ plants, an additionalcarboxylation reaction involving another enzyme, phosphoenol-pyruvatecarboxylase, is the primary carboxylation reaction. The first stablecarbon compound is a 4-carbon acid that is subsequently decarboxylated.The CO₂ thus released is refixed by the C₃ cycle. Examples of C₄ plantsare tropical grasses, corn, and sugar cane. Both C₄ and C₃ plantsexhibit a range of ¹³C/¹²C isotopic ratios, but typical values are about−7 to about −13 per mil for C₄ plants and about −19 to about −27 per milfor C₃ plants (see, e.g., Stuiver et al., Radiocarbon 19:355 (1977)).Coal and petroleum fall generally in this latter range. The ¹³Cmeasurement scale was originally defined by a zero set by Pee DeeBelemnite (PDB) limestone, where values are given in parts per thousanddeviations from this material. The “δ¹³C” values are expressed in partsper thousand (per mil), abbreviated, %, and are calculated as follows:δ¹³C(%)=[(¹³C/¹²C)_(sample)−(¹³C/¹²C)_(standard)]/(¹³C/¹²C)_(standard)×1000Since the PDB reference material (RM) has been exhausted, a series ofalternative RMs have been developed in cooperation with the IAEA, USGS,NIST, and other selected international isotope laboratories. Notationsfor the per mil deviations from PDB is δ¹³C. Measurements are made onCO₂ by high precision stable ratio mass spectrometry (IRMS) on molecularions of masses 44, 45, and 46. The compositions described herein includebioproducts produced by any of the methods described herein, including,for example, fatty aldehyde and alcohol products. Specifically, thebioproduct can have a δ¹³C of about −28 or greater, about −27 orgreater, −20 or greater, −18 or greater, −15 or greater, −13 or greater,−10 or greater, or −8 or greater. For example, the bioproduct can have aδ¹³C of about −30 to about −15, about −27 to about −19, about −25 toabout −21, about −15 to about −5, about −13 to about −7, or about −13 toabout −10. In other instances, the bioproduct can have a δ¹³C of about−10, −11, −12, or −12.3. Bioproducts, including the bioproducts producedin accordance with the disclosure herein, can also be distinguished frompetroleum based organic compounds by comparing the amount of ¹⁴C in eachcompound. Because ¹⁴C has a nuclear half-life of 5730 years, petroleumbased fuels containing “older” carbon can be distinguished frombioproducts which contain “newer” carbon (see, e.g., Currie, “SourceApportionment of Atmospheric Particles”, Characterization ofEnvironmental Particles, J. Buffle and H. P. van Leeuwen, Eds., 1 ofVol. I of the IUPAC Environmental Analytical Chemistry Series (LewisPublishers, Inc.) 3-74, (1992)).

The basic assumption in radiocarbon dating is that the constancy of ¹⁴Cconcentration in the atmosphere leads to the constancy of ¹⁴C in livingorganisms. However, because of atmospheric nuclear testing since 1950and the burning of fossil fuel since 1850, ¹⁴C has acquired a second,geochemical time characteristic. Its concentration in atmospheric CO₂,and hence in the living biosphere, approximately doubled at the peak ofnuclear testing, in the mid-1960s. It has since been gradually returningto the steady-state cosmogenic (atmospheric) baseline isotope rate(¹⁴C/¹²C) of about 1.2×10⁻¹², with an approximate relaxation “half-life”of 7-10 years. (This latter half-life must not be taken literally;rather, one must use the detailed atmospheric nuclear input/decayfunction to trace the variation of atmospheric and biospheric ¹⁴C sincethe onset of the nuclear age.) It is this latter biospheric ¹⁴C timecharacteristic that holds out the promise of annual dating of recentbiospheric carbon. ¹⁴C can be measured by accelerator mass spectrometry(AMS), with results given in units of “fraction of modern carbon”(f_(M)). f_(M) is defined by National Institute of Standards andTechnology (NIST) Standard Reference Materials (SRMs) 4990B and 4990C.As used herein, fraction of modern carbon (f_(M)) has the same meaningas defined by National Institute of Standards and Technology (NIST)Standard Reference Materials (SRMs) 4990B and 4990C, known as oxalicacids standards HOxI and HOxII, respectively. The fundamental definitionrelates to 0.95 times the ¹⁴C/¹²C isotope ratio HOxI (referenced to AD1950). This is roughly equivalent to decay-corrected pre-IndustrialRevolution wood. For the current living biosphere (plant material),f_(M) is approximately 1.1. This is roughly equivalent todecay-corrected pre-Industrial Revolution wood. For the current livingbiosphere (plant material), f_(M) is approximately 1.1.

The compositions described herein include bioproducts that can have anf_(M) ¹⁴C of at least about 1. For example, the bioproduct of thedisclosure can have an f_(M) ¹⁴C of at least about 1.01, an f_(M) ¹⁴C ofabout 1 to about 1.5, an f_(M) ¹⁴C of about 1.04 to about 1.18, or anf_(M) ¹⁴C of about 1.111 to about 1.124. Another measurement of ¹⁴C isknown as the percent of modern carbon (pMC). For an archaeologist orgeologist using ¹⁴C dates, AD 1950 equals “zero years old”. This alsorepresents 100 pMC. “Bomb carbon” in the atmosphere reached almost twicethe normal level in 1963 at the peak of thermo-nuclear weapons. Itsdistribution within the atmosphere has been approximated since itsappearance, showing values that are greater than 100 pMC for plants andanimals living since AD 1950. It has gradually decreased over time withtoday's value being near 107.5 pMC. This means that a fresh biomassmaterial, such as corn, would give a ¹⁴C signature near 107.5 pMC.Petroleum based compounds will have a pMC value of zero. Combiningfossil carbon with present day carbon will result in a dilution of thepresent day pMC content. By presuming 107.5 pMC represents the ¹⁴Ccontent of present day biomass materials and 0 pMC represents the ¹⁴Ccontent of petroleum based products, the measured pMC value for thatmaterial will reflect the proportions of the two component types. Forexample, a material derived 100% from present day soybeans would give aradiocarbon signature near 107.5 pMC. If that material was diluted 50%with petroleum based products, it would give a radiocarbon signature ofapproximately 54 pMC. A biologically based carbon content is derived byassigning “100%” equal to 107.5 pMC and “0%” equal to 0 pMC. Forexample, a sample measuring 99 pMC will give an equivalent biologicallybased carbon content of 93%. This value is referred to as the meanbiologically based carbon result and assumes all the components withinthe analyzed material originated either from present day biologicalmaterial or petroleum based material. A bioproduct comprising one ormore fatty aldehydes or alcohols as described herein can have a pMC ofat least about 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100.In other instances, a bioproduct described herein can have a pMC ofbetween about 50 and about 100; about 60 and about 100; about 70 andabout 100; about 80 and about 100; about 85 and about 100; about 87 andabout 98; or about 90 and about 95. In yet other instances, a bioproductdescribed herein can have a pMC of about 90, 91, 92, 93, 94, or 94.2.

Screening Fatty Alcohol Compositions Produced by Recombinant Host Cell

To determine if conditions are sufficient to allow expression, arecombinant host cell comprising a heterologous gene or a modifiednative gene is cultured, for example, for about 4, 8, 12, 24, 36, or 48hours. During and/or after culturing, samples can be obtained andanalyzed to determine if the fatty alcohol production level (titer,yield or productivity) is different than that of the corresponding wildtype parental cell which has not been modified. For example, the mediumin which the host cells were grown can be tested for the presence of adesired product. When testing for the presence of a product, assays,such as, but not limited to, TLC, HPLC, GC/FID, GC/MS, LC/MS, MS, can beused. Recombinant host cell strains can be cultured in small volumes(0.001 L to 1 L) of media in plates or shake flasks in order to screenfor altered fatty alcohol or fatty species production level. Oncecandidate strains or “hits” are identified at small scale, these strainsare cultured in larger volumes (1 L to 1000 L) of media in bioreactors,tanks, and pilot plants to determine the precise fatty alcohol or fattyspecies production level. These large volume culture conditions are usedby those skilled in the art to optimize the culture conditions to obtaindesired fatty alcohol or fatty species production.

Utility of Fatty Aldehyde and Fatty Alcohol Compositions

Aldehydes are used to produce many specialty chemicals. For example,aldehydes are used to produce polymers, resins (e.g., Bakelite), dyes,flavorings, plasticizers, perfumes, pharmaceuticals, and otherchemicals, some of which may be used as solvents, preservatives, ordisinfectants. In addition, certain natural and synthetic compounds,such as vitamins and hormones, are aldehydes, and many sugars containaldehyde groups. Fatty aldehydes can be converted to fatty alcohols bychemical or enzymatic reduction. Fatty alcohols have many commercialuses. Worldwide annual sales of fatty alcohols and their derivatives arein excess of U.S. $1 billion. The shorter chain fatty alcohols are usedin the cosmetic and food industries as emulsifiers, emollients, andthickeners. Due to their amphiphilic nature, fatty alcohols behave asnonionic surfactants, which are useful in personal care and householdproducts, such as, for example, detergents. In addition, fatty alcoholsare used in waxes, gums, resins, pharmaceutical salves and lotions,lubricating oil additives, textile antistatic and finishing agents,plasticizers, cosmetics, industrial solvents, and solvents for fats. Thedisclosure also provides a surfactant composition or a detergentcomposition comprising a fatty alcohol produced by any of the methodsdescribed herein. One of ordinary skill in the art will appreciate that,depending upon the intended purpose of the surfactant or detergentcomposition, different fatty alcohols can be produced and used. Forexample, when the fatty alcohols described herein are used as afeedstock for surfactant or detergent production, one of ordinary skillin the art will appreciate that the characteristics of the fatty alcoholfeedstock will affect the characteristics of the surfactant or detergentcomposition produced. Hence, the characteristics of the surfactant ordetergent composition can be selected for by producing particular fattyalcohols for use as a feedstock. A fatty alcohol-based surfactant and/ordetergent composition described herein can be mixed with othersurfactants and/or detergents well known in the art. In someembodiments, the mixture can include at least about 10%, at least about15%, at least about 20%, at least about 30%, at least about 40%, atleast about 50%, at least about 60%, or a range bounded by any two ofthe foregoing values, by weight of the fatty alcohol. In other examples,a surfactant or detergent composition can be made that includes at leastabout 5%, at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, or a range bounded by any two of the foregoing values,by weight of a fatty alcohol that includes a carbon chain that is 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 carbons in length.Such surfactant or detergent compositions also can include at least oneadditive, such as a microemulsion or a surfactant or detergent fromnonmicrobial sources such as plant oils or petroleum, which can bepresent in the amount of at least about 5%, at least about 10%, at leastabout 15%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,or a range bounded by any two of the foregoing values, by weight of thefatty alcohol. The disclosure is further illustrated by the followingexamples. The examples are provided for illustrative purposes only. Theyare not to be construed as limiting the scope or content of thedisclosure in any way.

EXAMPLES Example 1

Production Host Modifications—Attenuation of Acyl-CoA Dehydrogenase

This example describes the construction of a genetically engineered hostcell wherein the expression of a fatty acid degradation enzyme isattenuated. The fadE gene of Escherichia coli MG1655 (an E. coli Kstrain) was deleted using the Lambda Red (also known as the Red-DrivenIntegration) system described by Datsenko et al., Proc. Natl. Acad. Sci.USA 97: 6640-6645 (2000), with the following modifications:

The following two primers were used to create the deletion of fadE:

Del-fadE- (SEQ ID NO: 9)F5′-AAAAACAGCAACAATGTGAGCTTTGTTGTAATTATATTGTAAACATATTGATTCCGGGGATCCGTCGACC; and Del-fadE- (SEQ ID NO: 10)R5′-AAACGGAGCCTTTCGGCTCCGTTATTCATTTACGCGGCTTCAACTTTCCTGTAGGCTGGAGCTGCTTC

The Del-fadE-F and Del-fadE-R primers were used to amplify the kanamycinresistance (KmR) cassette from plasmid pKD13 (described by Datsenko etal., supra) by PCR. The PCR product was then used to transformelectrocompetent E. coli MG1655 cells containing pKD46 (described inDatsenko et al., supra) that had been previously induced with arabinosefor 3-4 hours. Following a 3-hour outgrowth in a super optimal brothwith catabolite repression (SOC) medium at 37° C., the cells were platedon Luria agar plates containing 50 μg/mL of Kanamycin. Resistantcolonies were identified and isolated after an overnight incubation at37° C. Disruption of the fadE gene was confirmed by PCR amplificationusing primers fadE-L2 and fadE-R1, which were designed to flank the E.coli fadE gene.

The fadE deletion confirmation primers were:

fadE-L2 (SEQ ID NO: 11) 5′-CGGGCAGGTGCTATGACCAGGAC; and fadE-R1 (SEQ IDNO: 12) 5′-CGCGGCGTTGACCGGCAGCCTGG

After the fadE deletion was confirmed, a single colony was used toremove the KmR marker using the pCP20 plasmid as described by Datsenkoet al., supra. The resulting MG1655 E. coli strain with the fadE genedeleted and the KmR marker removed was named E. coli MG1655 ΔfadE, or E.coli MG 1655 D1. Fatty acid derivative (“Total Fatty Species”)production by the MG1655 E. coli strain with the fadE gene deleted wascompared to fatty acid derivative production by E. coli MG1655. Cellswere transformed with production plasmid pDG109 (pCL1920_P_(TRC)_(_)carBopt_12H08_alrAadp1_fabB[A329G]_fadR) and fermented in glucoseminimal media. The data presented in FIG. 5 shows that deletion of thefadE gene did not affect fatty acid derivative production.

Example 2

Increased Flux Through the Fatty Acid Synthesis Pathway—Acetyl CoACarboxylase Mediated

The main precursors for fatty acid biosynthesis are malonyl-CoA andacetyl-CoA (FIG. 1). It has been suggested that these precursors limitthe rate of fatty acid biosynthesis (FIG. 2) in E. coli. In thisexample, synthetic acc operons [Corynebacterium glutamicum accABCD(±birA)] were overexpressed and the genetic modifications led toincreased acetyl-coA and malonyl-CoA production in E. coli. In oneapproach, in order to increase malonyl-CoA levels, an acetyl-CoAcarboxylase enzyme complex from Corynebacterium glutamicum (C.glutamicum) was overexpressed in E. coli. Acetyl-CoA carboxylase (acc)consists of four discrete subunits, accA, accB, accC and accD (FIG. 3).The advantage of C. glutamicum acc is that two subunits are expressed asfusion proteins, accCB and accDA, respectively, which facilitates itsbalanced expression. Additionally, C. glutamicum birA, whichbiotinylates the accB subunit (FIG. 3) was overexpressed. Example 3describes co-expression of acc genes together with entire fab operons.

Example 3

Increased Flux Through the Fatty Acid Synthesis Pathway—iFABs

Fatty Acid Derivative Production:

Strategies to increase the flux through the fatty acid synthesis pathwayin recombinant host cells include both overexpression of native E. colifatty acid biosynthesis genes and expression of exogenous fatty acidbiosynthesis genes from different organisms in E. coli. In this study,fatty acid biosynthesis genes from different organisms were combined inthe genome of E. coli DV2. Sixteen strains containing iFABs 130-145 wereevaluated. The detailed structure of iFABs 130-145 is presented in iFABsTable 1, below.

TABLE 1 Components found in iFABs 130-145. Abbreviation Full DescriptionSt_fabD Salmonella typhimurium fabD gene nSt_fabH Salmonella typhimuriumfabH gene with the native RBS sSt_fabH Salmonella typhimurium fabH genewith a synthetic RBS Cac_fabF Clostridium acetobutylicum (ATCC824) fabFgene St_fabG Salmonella typhimurium fabG gene St_fabA Salmonellatyphimurium fabA gene St_fabZ Salmonella typhimurium fabZ gene BS_fabIBacillus subtilis fabI gene BS_FabL Bacillus subtilis fabL gene Vc_FabVVibrio chorlerae fabV gene Ec_FabI Escherichia coli fabI gene

Each “iFAB” included various fab genes in the following order: 1) anenoyl-ACP reductase (BS_fabI, BS_FabL, Vc_FabV, or Ec_FabI); 2) ab-ketoacyl-ACP synthetase III (St_fabH); 3) a malonyl-CoA-ACPtransacylase (St_fabD); 4) a b-ketoacyl-ACP reductase (St_fabG); 5) a3-hydroxy-acyl-ACP dehydratase (St_fabA or St_fabZ); 6) a b-ketoacyl-ACPsynthetase II (Cac_fabF). Note that St_fabA also has trans-2,cis-3-decenoyl-ACP isomerase activity (ref) and that Cac_fabF hasb-ketoacyl-ACP synthetase II and b-ketoacyl-ACP synthetase I activities(Zhu et al., BMC Microbiology 9:119 (2009)). See Table 2, below for thespecific composition of iFABs 130-145. See FIGS. 7A and B which providediagrammatic depiction of the iFAB138 locus, including a diagram ofcat-loxP-T5 promoter integrated in front of FAB138 (7A); and a diagramof iT5_138 (7B).

TABLE 2 Composition of iFABs 130-145. ifab BS_fabl BS_fabL Vc_fabVEc_fabl nSt_fabH sSt_fabH St_fabD St_fabG St_fabA St_fabZ Cac_fabFifab130 1 0 0 0 1 0 1 1 1 0 1 ifab131 1 0 0 0 1 0 1 1 0 1 1 ifab132 1 00 0 0 1 1 1 1 0 1 ifab133 1 0 0 0 0 1 1 1 0 1 1 ifab134 0 1 0 0 1 0 1 11 0 1 ifab135 0 1 0 0 1 0 1 1 0 1 1 ifab136 0 1 0 0 0 1 1 1 1 0 1Ifab137 0 1 0 0 0 1 1 1 0 1 1 ifab138 0 0 1 0 1 0 1 1 1 0 1 ifab139 0 01 0 1 0 1 1 0 1 1 ifab140 0 0 1 0 0 1 1 1 1 0 1 ifab141 0 0 1 0 0 1 1 10 1 1 ifab142 0 0 0 1 1 0 1 1 1 0 1 ifab143 0 0 0 1 1 0 1 1 0 1 1ifab144 0 0 0 1 0 1 1 1 1 0 1 ifab145 0 0 0 1 0 1 1 1 0 1 1

The plasmid pCL_P_(trc) _(_)tesA was transformed into each of thestrains and a fermentation was run in FA2 media with 20 hours frominduction to harvest at both 32° C. and 37° C. Data for production ofTotal Fatty Species from duplicate plate screens is shown in FIGS. 6Aand 6B. From this library screen the best construct was determined to beDV2 with iFAB138. The iFAB138 construct was transferred into strain D178to make strain EG149. This strain was used for further engineering. Thesequence of iFAB138 in the genome of EG149 is presented as SEQ ID NO:13.Table 3 presents the genetic characterization of a number of E. colistrains into which plasmids containing the expression constructsdescribed herein were introduced as described below. These strains andplasmids were used to demonstrate the recombinant host cells, cultures,and methods of certain embodiments of the present disclosure. Thegenetic designations in Table 3 are standard designations known to thoseof ordinary skill in the art.

TABLE 3 Genetic Characterization of E. coli strains Strain GeneticCharacterization DV2 MG1655 F-, λ-, ilvG-, rfb-50, rph-1, ΔfhuA::FRT,ΔfadE::FRT DV2.1 DV2 fabB::fabB[A329V] D178 DV2.1 entD::FRT_P_(T5) _(—)entD EG149 D178 ΔinsH-11::P_(LACUV5)-iFAB138 V642 EG149 rph+ SL313 V642lacIZ::P_(A1) _(—) ′tesA/pDG109 V668 V642 ilvG⁺ LC397 V668lacIZ::P_(TRC) _(—) ′tesA(var)_kan SL571 V668 lacIZ:: P_(TRC) _(—)′tesA(var)_FRT LC942 SL571 attTn7::P_(TRC) _(—) ′tesA(var) DG16LC942/pLC56 V940 LC397/pV171.1 D851 SL571 yijP::Tn5-cat/pV171.1Plasmids: pDG109, pLC56 and pV171.1 are pCL_P_(trc) _(—)carB_tesA_alrA_fabB_fadR operon with variable expression of carB andtesA. iFAB138 is SEQ ID NO: 13.

Example 4

Increasing the Amount of Free Fatty Acid (FFA) Product by Repairing theRph and ilvG Mutations

The ilvG and rph mutations were corrected in this strain resulting inhigher production of FFA. Strains D178, EG149 and V668 (Table 3) weretransformed with pCL_P_(trc) _(_)tesA. Fermentation was run at 32° C. inFA2 media for 40 hours to compare the FFA production of strains D178,EG149, and V668 with pCL_P_(trc) _(_)tesA. Correcting the rph and ilvGmutations resulted in a 116% increase in the FFA production of the basestrain with pCL_P_(trc) _(_)tesA. As seen in FIG. 8, V668/pCL_P_(trc)_(_)tesA produces more FFA than the D178/pCL_P_(trc) _(_)tesA, or theEG149/pCL_P_(trc) _(_)tesA control. Since FFA is a precursor to the LS9products, higher FFA production is a good indicator that the new straincan produce higher levels of LS9 products. Fermentation and extractionwas run according to a standard FALC fermentation protocol exemplifiedby the following.

A frozen cell bank vial of the selected E. coli strain was used toinoculate 20 mL of LB broth in a 125 mL baffled shake flask containingspectinomycin antibiotic at a concentration of 115 μg/mL. This shakeflask was incubated in an orbital shaker at 32° C. for approximately sixhours, then 1.25 mL of the broth was transferred into 125 mL of low PFA2 seed media (2 g/L NH₄Cl, 0.5 g/L NaCl, 3 g/L KH₂PO₄, 0.25 g/LMgSO₄-7H2O, 0.015 g/L mM CaCl₂-2H2O, 30 g/L glucose, 1 mL/L of a traceminerals solution (2 g/L of ZnCl₂.4H₂O, 2 g/L of CaCl₂.6H₂O, 2 g/L ofNa₂MoO₄.2H₂O, 1.9 g/L of CuSO₄.5H₂O, 0.5 g/L of H₃BO₃, and 10 mL/L ofconcentrated HCl), 10 mg/L of ferric citrate, 100 mM of Bis-Tris buffer(pH 7.0), and 115 μg/mL of spectinomycin), in a 500 mL baffledErlenmeyer shake flask, and incubated on a shaker overnight at 32° C.100 mL of this low P FA2 seed culture was used to inoculate a 5 LBiostat Aplus bioreactor (Sartorius BBI), initially containing 1.9 L ofsterilized F1 bioreactor fermentation medium. This medium is initiallycomposed of 3.5 g/L of KH₂PO₄, 0.5 g/L of (NH₄)₂SO₄, 0.5 g/L of MgSO₄heptahydrate, 10 g/L of sterile filtered glucose, 80 mg/L ferriccitrate, 5 g/L Casamino acids, 10 mL/L of the sterile filtered traceminerals solution, 1.25 mL/L of a sterile filtered vitamin solution(0.42 g/L of riboflavin, 5.4 g/L of pantothenic acid, 6 g/L of niacin,1.4 g/L of pyridoxine, 0.06 g/L of biotin, and 0.04 g/L of folic acid),and the spectinomycin at the same concentration as utilized in the seedmedia. The pH of the culture was maintained at 6.9 using 28% w/v ammoniawater, the temperature at 33° C., the aeration rate at 1 lpm (0.5v/v/m), and the dissolved oxygen tension at 30% of saturation, utilizingthe agitation loop cascaded to the DO controller and oxygensupplementation. Foaming was controlled by the automated addition of asilicone emulsion based antifoam (Dow Corning 1410).

A nutrient feed composed of 3.9 g/L MgSO₄ heptahydrate and 600 g/Lglucose was started when the glucose in the initial medium was almostdepleted (approximately 4-6 hours following inoculation) under anexponential feed rate of 0.3 hr⁻¹ to a constant maximal glucose feedrate of 10-12 g/L/hr, based on the nominal fermentation volume of 2 L.Production of fatty alcohol in the bioreactor was induced when theculture attained an OD of 5 AU (approximately 3-4 hours followinginoculation) by the addition of a 1M IPTG stock solution to a finalconcentration of 1 mM. The bioreactor was sampled twice per daythereafter, and harvested approximately 72 hours following inoculation.A 0.5 mL sample of the well-mixed fermentation broth was transferredinto a 15 mL conical tube (VWR), and thoroughly mixed with 5 mL of butylacetate. The tube was inverted several times to mix, then vortexedvigorously for approximately two minutes. The tube was then centrifugedfor five minutes to separate the organic and aqueous layers, and aportion of the organic layer transferred into a glass vial for gaschromatographic analysis.

Example 5

Increased Production of Fatty Alcohol by Transposon Mutagenesis—yijP

To improve the titer, yield, productivity of fatty alcohol production byE. coli, transposon mutagenesis and high-throughput screening wascarried out and beneficial mutations were sequenced. A transposoninsertion in the yijP strain was shown to improve the strain's fattyalcohol yield in both shake flask and fed-batch fermentations. The SL313strain produces fatty alcohols. The genotype of this strain is providedin Table 3. Transposon clones were then subjected to high-throughputscreening to measure production of fatty alcohols. Briefly, colonieswere picked into deep-well plates containing LB, grown overnight,inoculated into fresh LB and grown for 3 hours, inoculated into freshFA2.1 media, grown for 16 hours, then extracted using butyl acetate. Thecrude extract was derivatized with BSTFA(N,O-bis[Trimethylsilyl]trifluoroacetamide) and analyzed using GC/FID.Spectinomycin (100 mg/L) was included in all media to maintain selectionof the pDG109 plasmid. Hits were selected by choosing clones thatproduced a similar total fatty species as the control strain SL313, butthat had a higher percent of fatty alcohol species and a lower percentof free fatty acids than the control. Strain 68F11 was identified as ahit and was validated in a shake flask fermentation using FA2.1 media. Acomparison of transposon hit 68F11 to control strain SL313 indicatedthat 68F11 produces a higher percentage of fatty alcohol species thanthe control, while both strains produce similar titers of total fattyspecies. A single colony of hit 68F11, named LC535, was sequenced toidentify the location of the transposon insertion. Briefly, genomic DNAwas purified from a 10 mL overnight LB culture using the kit ZRFungal/Bacterial DNA MiniPrep™ (Zymo Research Corporation, Irvine,Calif.) according to the manufacturer's instructions. The purifiedgenomic DNA was sequenced outward from the transposon using primersinternal to the transposon:

DG150 (SEQ ID NO: 14) 5′-GCAGTTATTGGTGCCCTTAAACGCCTGGTTGCTACGCCTG-3′DG131 (SEQ ID NO: 15) 5′-GAGCCAATATGCGAGAACACCCGAGAA-3′

Strain LC535 was determined to have a transposon insertion in the yijPgene (FIG. 18). yijP encodes a conserved inner membrane protein whosefunction is unclear. The yijP gene is in an operon and co-transcribedwith the ppc gene, encoding phosphoenolpyruvate carboxylase, and theyijO gene, encoding a predicted DNA-binding transcriptional regulator ofunknown function. Promoters internal to the transposon likely haveeffects on the level and timing of transcription of yijP, ppc and yijO,and may also have effects on adjacent genes frwD, pflC, pfld, and argE.Promoters internal to the transposon cassette are shown in FIG. 18, andmay have effects on adjacent gene expression. Strain LC535 was evaluatedin a fed-batch fermentation on two different dates. Both fermentationsdemonstrated that LC535 produced fatty alcohols with a higher yield thancontrol SL313, and the improvement was 1.3-1.9% absolute yield based oncarbon input. The yijP transposon cassette was further evaluated in adifferent strain V940, which produces fatty alcohol at a higher yieldthan strain SL313. The yijP::Tn5-cat cassette was amplified from strainLC535 using primers:

LC277 (SEQ ID NO: 16) 5′-CGCTGAACGTATTGCAGGCCGAGTTGCTGCACCGCTCCCGCCAGGCAG-3′ LC278 (SEQ ID NO: 17)5′-GGAATTGCCACGGTGCGGCAGGCTCCATACGCGAGGCCAGGTTAT CCAACG-3′

This linear DNA was electroporated into strain SL571 and integrated intothe chromosome using the lambda red recombination system. Colonies werescreened using primers outside the transposon region:

DG407 (SEQ ID NO: 18) 5′-AATCACCAGCACTAAAGTGCGCGGTTCGTTACCCG-3′ DG408(SEQ ID NO: 19) 5′-ATCTGCCGTGGATTGCAGAGTCTATTCAGCTACG-3′

A colony with the correct yijP transposon cassette (FIG. 9) wastransformed with the production plasmid pV171.1 to produce strain D851.D851 (V940 yijP::Tn5-cat) was tested in a shake-flask fermentationagainst isogenic strain V940 that does not contain the yijP transposoncassette. The result of this fermentation showed that the yijPtransposon cassette confers production of a higher percent of fattyalcohol by the D851 strain relative to the V940 strain and producessimilar titers of total fatty species as the V940 control strain. StrainD851 was evaluated in a fed-batch fermentation on two different dates.Data from these fermentations is shown in Table 4 which illustrates thatin 5-liter fed-batch fermentations, strains with the yijP::Tn5-cattransposon insertion had an increased total fatty species (“FAS”) yieldand an increase in percent fatty alcohol (“FALC”). “Fatty Species”include FALC and FFA.

TABLE 4 Effect of yijp transposon insertion on titer and yield of FASand FALC Strain FAS Titer FAS Yield Percent FALC FALC Yield V940 68 g/L18.70% 95.00% 17.80% D851 70 g/L 19.40% 96.10% 18.60% V940 64 g/L 18.40%91.90% 16.90% D851 67 g/L 19.00% 94.00% 17.80%

Tank Fermentation Method:

To assess production of fatty acid esters in tank a glycerol vial ofdesired strain was used to inoculate 20 mL LB+spectinomycin in shakeflask and incubated at 32° C. for approximately six hours. 4 mL of LBculture was used to inoculate 125 mL Low PFA Seed Media (below), whichwas then incubated at 32° C. shaker overnight. 50 mL of the overnightculture was used to inoculate 1 L of Tank Media. Tanks were run at pH7.2 and 30.5° C. under pH stat conditions with a maximum feed rate of 16g/L/hr (glucose or methanol).

TABLE 5 Low P FA Seed Media Component Concentration NH4Cl 2 g/L NaCl 0.5g/L KH2PO4 1 g/L MgSO4—7H2O 0.25 g/L CaCl2—2H2O 0.015 g/L Glucose 20 g/LTM2 Trace Minerals solution 1 mL/L Ferric citrate 10 mg/L Bis Trisbuffer (pH 7.0) 100 mM Spectinomycin 115 mg/L

TABLE 6 Tank Media Component Concentration (NH4)2SO4 0.5 g/L KH2PO4 3.0g/L Ferric Citrate 0.034 g/L TM2 Trace Minerals Solution 10 mL/LCasamino acids 5 g/L Post sterile additions MgSO4—7H2O 2.2 g/L TraceVitamins Solution 1.25 mL/L Glucose 5 g/L Inoculum 50 mL/L

Example 6

Addition of an N-Terminal 60 bp Fusion Tag to CarB (CarB60)

There are many ways to increase the solubility, stability, expression orfunctionality of a protein. In one approach to increasing the solubilityof CarB, a fusion tag could be cloned before the gene. In anotherapproach increase the expression of CarB, the promoter or ribosomebinding site (RBS) of the gene could be altered. In this study, carB(SEQ ID NO: 7) was modified by addition of an N-terminal 60 bp fusiontag. To generate the modified protein (referred to herein as “CarB60”),carB was first cloned into the pET15b vector using primers:

(SEQ ID NO: 20) 5′-GCAATTCCATATGACGAGCGATGTTCACGA-3′; and (SEQ ID NO:21) 5′-CCGCTCGAGTAAATCAGACCGAACTCGCG.

The pET15b—carB construct contained 60 nucleotides directly upstream ofthe carB gene:

(SEQ ID NO: 22) 5′-ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCAT

The fusion tag version of carB was renamed carB60. The pET15b_carB60 wasthen digested using restriction enzymes NcoI and HindIII and subclonedinto the pCL1920-derived vector OP80 which was cut with the sameenzymes. This plasmid was transformed into strain V324 (MG1655ΔfadE::FRT ΔfhuA::FRT fabB::A329V entD::T5-entD lacIZ::P_(TRC)-'TesA) toevaluate FALC production. Strains were fermented according to a standardprocedure (summarized below) and the total fatty species titer and totalfatty alcohol titer were quantified. FIG. 10 shows that CarB60 increasesfatty alcohol titers and therefore the CarB60 enzyme has higher totalcellular activity than CarB when expressed from a multicopy plasmid.

To assess production of fatty alcohols in production strains,transformants were grown in 2 ml of LB broth supplemented withantibiotics (100 mg/L) at 37° C. After overnight growth, 40 ul ofculture was transferred into 2 ml of fresh LB supplemented withantibiotics. After 3 hours of growth, 2 ml of culture were transferredinto a 125 mL flask containing 20 ml of M9 medium with 3% glucosesupplemented with 20 μl trace mineral solution, 10 μg/L iron citrate, 1μg/L thiamine, and antibiotics (FA2 media). When the OD₆₀₀ of theculture reached 1.0, 1 mM of IPTG was added to each flask. After 20hours of growth at 37° C., 400 μL samples from each flask were removedand fatty alcohols extracted with 400 μL butyl acetate. To furtherunderstand the mechanism of the improved CarB activity, CarB60 waspurified from strain D178 which does not contain 'TesA (MG1655ΔfadE::FRT ΔfhuA::FRT fabB::A329V entD::P_(T5)-entD). Briefly,pCL1920_carB60 was transformed into strain D178, which has beenengineered for fatty alcohol production, and fermentation was carriedout at 37° C. in FA-2 medium supplemented with spectinomycin (100μg/ml). When the culture OD₆₀₀ reached 1.6, cells were induced with 1 mMisopropyl-β-D-thiogalactopyranoside (IPTG) and incubated for anadditional 23 h at 37° C. For purification of CarB60, the cells wereharvested by centrifugation for 20 min at 4° C. at 4,500 rpm. Cell paste(10 g) was suspended in 12 ml of BugBuster MasterMix (Novagen) andprotease inhibitor cocktail solution. The cells were disrupted by FrenchPress and the resulting homogenate was centrifuged at 10,000 rpm toremove cellular debris. Ni-NTA was added to the resulting mixture, andthe suspension was swirled at 4° C. at 100 rpm for 1 hour on a rotaryshaker. The slurry was poured into a column, and the flow-through wascollected. The Ni-NTA resin was washed with 10 mM imidazole in 50 mMsodium phosphate buffer pH 8.0 containing 300 mM NaCl, and furtherwashed with 20 mM imidazole in 50 mM sodium phosphate buffer pH 8.0containing 300 mM NaCl. The CarB60 protein was eluted with 250 mMimidazole in 50 mM sodium phosphate buffer pH 8.0 containing 300 mMNaCl, and analyzed by SDS-PAGE. The protein was dialyzed against 20%(v/v) glycerol in 50 sodium phosphate buffer pH 7.5 yieldingapproximately 10 mg of CarB60 per liter of culture. The protein wasflash frozen and stored at −80° C. until needed.

The CarB60 protein was abundantly expressed from a multicopy plasmid.Additional SDS-PAGE analysis showed that expression of CarB60 was higherthan CarB. The higher expression level of CarB60 suggested that thecarB60 gene integrated into the E. coli chromosome would produce moreprotein than the carB gene in the same location. To test thishypothesis, the carB60 gene was integrated into the E. coli chromosome.Briefly, the carB60 gene was first amplified from pCL_carB60 usingforward primer:

(SEQ ID NO: 23) 5′-ACGGATCCCCGGAATGCGCAACGCAATTAATGTaAGTTAGCGC-3′;and reverse primer:

(SEQ ID NO: 24) 5′-TGCGTCATCGCCATTGAATTCCTAAATCAGACCGAACTCGCGCAG G-3′.

A second PCR product was amplified from vector pAH56 using forwardprimer:

(SEQ ID NO: 25) 5′-ATTCCGGGGATCCGTCGACC-3′;and reverse primer:

(SEQ ID NO: 26) 5′-AATGGCGATGACGCATCCTCACG-3′

This fragment contains a kanamycin resistance cassette, λattP site, andγR6k origin of replication. The two PCR products were joined using theInFusion kit (Clontech) to create plasmid pSL116-126. A fatty alcoholproduction strain containing an integrated form of 'TesA12H08 and ahelper plasmid pINT was transformed with either pSL116-126 containingthe carB60 gene or plasmid F27 containing the carB gene. These strainswere fermented in FA2 media according to standard procedures forshake-flask fermentations, as described above. To characterize andquantify the fatty alcohols and fatty acid esters, gas chromatography(“GC”) coupled with flame ionization (“FID”) detection was used. Thecrude extract was derivatized with BSTFA(N,O-bis[Trimethylsilyl]trifluoroacetamide) and analyzed using a GC/FID.Quantification was carried out by injecting various concentrations ofthe appropriate authentic references using the GC method described aboveas well as assays including, but not limited to, gas chromatography(GC), mass spectroscopy (MS), thin layer chromatography (TLC),high-performance liquid chromatography (HPLC), liquid chromatography(LC), GC coupled with a flame ionization detector (GC-FID), GC-MS, andLC-MS, can be used. When testing for the expression of a polypeptide,techniques such as Western blotting and dot blotting may be used.

The results of the fermentation after 20 hours are shown in FIG. 11. Thetotal fatty product titers of the two strains are similar (2.4 g/L totalfatty species), however integrated CarB60 converts a greater fraction ofC12 and C14 chain length free fatty acids into fatty alcohols, comparedto CarB without the N-terminal tag. These data suggest that cellsexpressing CarB60 have a higher total cellular carboxylic acid reductaseactivity, and can convert more FFA into fatty alcohols. Thus, carB60when integrated in the chromosome is an improved carB template thatprovides desired activity for evolving carB gene to identify improvedcarB variants.

Example 7

Generation of CarB Mutants

The CarB enzyme is a rate-limiting step in the production of fattyalcohols under certain process conditions. To produce fatty alcoholseconomically, efforts were made to increase the activity of the CarBenzyme.

Error Prone PCR Library Screen:

Random mutagenesis using error prone PCR was performed under conditionswhere the copying fidelity of the DNA polymerase is low. The mutagenizednucleic acids were cloned into a vector, and error-prone PCR followed byhigh-throughput screening was done to find beneficial mutations thatincrease conversion of free fatty acids to fatty alcohols (as detailedbelow). Important residues were further mutated to other amino acids. Anumber of single amino acid mutations and combinations of mutationsincreased the fraction of fatty species that are converted to fattyalcohols. Briefly, random mutations were generated in the carB60opt geneby error-prone PCR using the Genemorph II kit (Stratagene). Mutationswere generated in only one of two domains of carB60opt separately, tofacilitate cloning Library 1 contained the first 759 residues ofcarB60opt and was generated by error-prone PCR using primers:

HZ117 (SEQ ID NO: 27) 5′-ACGGAAAGGAGCTAGCACATGGGCAGCAGCCATCATCAT-3′; andDG264 (SEQ ID NO: 28) 5′-GTAAAGGATGGACGGCGGTCACCCGCC-3′.The vector for Library 1 was plasmid pDG115 digested with enzymes NheIand PshAI. Library 2 contained the last 435 residues of carB60opt andwas generated by error-prone PCR using primers:

DG263 (SEQ ID NO: 29) 5′-CACGGCGGGTGACCGCCGTCCATCC-3′; and HZ118 (SEQ IDNO: 30) 5′-TTAATTCCGGGGATCCCTAAATCAGACCGAACTCGCGCAGGTC-3′.

The vector for Library 2 was plasmid pDG115 digested with enzymes PshAIand BamHI. The error-prone inserts were cloned into the vectors usingInFusion Advantage (Clontech) and passaged through cloning strain NEBTurbo (New England Biolabs). The libraries were then transformed intostrain EG442 (EG149 Tn7::P_(TRC)-ABR lacIZ::P_(T5O)-ABR). Error-pronecarB60opt clones were then subjected to high-throughput screening tomeasure production of fatty alcohols. Briefly, colonies were picked intodeep-well plates containing LB, grown overnight, inoculated into freshLB and grown for 3 hours, inoculated into fresh FA-2.1 media, grown for16 hours, then extracted using butyl acetate. The crude extract wasderivatized with BSTFA (N,O-bis[Trimethylsilyl]trifluoroacetamide) andanalyzed using a standard GC/FID method. Spectinomycin (100 mg/L) wasincluded in all media to maintain selection of the pDG115 plasmid. Hitswere selected by choosing clones that produced a smaller total freefatty acid titer and a larger total fatty alcohol titer compared to thecontrol strain. To compare hits from different fermentation screens, theconversion of free fatty acids to fatty alcohols was normalized bycalculating a normalized free fatty acid percentage NORM FFA=MutantPercent FFA/Control Percent FFA where “Percent FFA” is the total freefatty acid species titer divided by the total fatty species titer. Hitswere subjected to further verification using shake-flask fermentations,as described below.

Hits were sequenced to identify the beneficial mutations. Sequencing wasperformed by colony PCR of the entire carB60opt gene using primers

SL59 (SEQ ID NO: 31) 5′-CAGCCGTTTATTGCCGACTGGATG-3′; and EG479 (SEQ IDNO: 32) 5′-CTGTTTTATCAGACCGCTTCTGCGTTC-3′,and sequenced using primers internal to the carB60opt enzyme.

The beneficial mutations that improved the CarB60opt enzyme are shown inTable 7. The normalized free fatty acid (NORM FFA) column indicates theimprovement in the enzyme, with lower values indicating the bestimprovement. “Well #” indicates the primary screening well that thismutation was found in. All residue numbers refer to the CarB proteinsequence, which does not include the 60 bp tag. Mutations indicated withthe prefix “Tag:” indication mutations in the 60 bp/20 residueN-terminal tag.

TABLE 7 Beneficial Mutations in the CarB Enzyme Identified During Error-Prone Screening (TAG Mutations Removed) Well # Norm FFA MissenseMutations Silent Mutations 131B08 70.50% L799M V810F S927R M1062L A1158VF1170I CCG1116CCT 20C07 71.80% A535S 65B02 74.70% M930R ACC867ACA 54B1076.30% L80Q 7231M F288L A418T V530M A541V G677D P712A 67E1 78.20% D750GR827C D986G G1026D P1149S GCA1031GCT GTC1073GTT 65C03 78.90% V926AATT941ATA 12C10 80.30% V46I 66E08 80.10% V926A 70F02 80.90% D750G R827CD986G G1026D P1149S GCA1031GCT GTC1073GTT 07D01 82.40% E20K V191A 66G0982.40% R827C L1128S ACG780ACA CTG923TTG 25H02 83.50% F288S 06C01 85.10%V46I 06C01 05D02 85.20% T396S CCG477CCT 124E03 86.00% R827C L1128SACG780ACA CTG923TTG 17A04 86.20% A574T GCA237GCT ACC676ACT GCC529GCT132C08 87.00% M1062T R1080H TTG830TTA TAC834TAT 72C09 87.30% P809LM1062V 10F02 87.70% E636K 71H03 88.10% R827C L1128S ACG780ACA CTG923TTG38G04 88.90% D143E A612T GCA181GCG 42F08 90.20% T90M CTG186CTT 66C0490.30% L1128S 18C03 90.40% Q473L 12E02 90.60% D19N S22N R87H L416SCCG167CCA 28B09 91.10% E28K H212N Q473L CCG122CCA ACG178ACA CTG283TTGCTG340CTA ACC401ACT GCA681GCG 103E09 92.20% E936K P1134R CGT829CGGCTG1007CTA 03E09 93.20% M259I 74G11 93.80% I870V S927I S985I I1164FGTG1000GTC 46C01 95.60% D18V D292N

Saturation Mutagenesis (Combo 1 and 2 Library Generated):

Amino acid positions deemed beneficial for fatty alcohol productionfollowing error-prone PCR were subjected to further mutagenesis. Primerscontaining the degenerate nucleotides NNK or NNS were used to mutatethese positions to other amino acids. The resulting “saturationmutagenesis libraries” were screened as described above for the errorprone libraries, and hits were identified that further improved fattyalcohol conversion (a smaller total free fatty acid titer and a largertotal fatty alcohol titer compared to the parent “control” strain).Single amino acid/codon changes in nine different positions that improvethe production of fatty alcohols are shown in Table 8. Hits weresubjected to further verification using shake-flask fermentations, asdescribed herein.

TABLE 8 Beneficial Mutations in the CarB Enzyme Identified During AminoAcid Saturation Mutagenesis WT Amino Mutant Acid WT Codon Amino AcidMutant Codon Norm FFA E20 GAG F TTC 92.20% L CTG 94.50% L TTG 96.20% RCGC 86.50% S TCG 87.40% V GTG 86.00% V GTC 85.30% Y TAC 88.80% V191 GTCA GCC 88.70% S AGT 98.00% F288 TTT G GGG 70.30% R AGG 77.20% S TCT85.60% S AGC 79.60% Q473 CAA A GCG 89.50% F TTC 89.10% H CAC 84.10% IATC 77.20% K AAG 90.30% L CTA 90.10% M ATG 89.00% R AGG 88.00% V GTG89.20% W TGG 84.50% Y TAC 86.00% A535 GCC A TCC 71.80% R827 CGC A GCC93.20% C TGT 87.90% C TGC 83.20% V926 GTT A GCT 78.10% A GCG 66.30% AGCC 69.50% E GAG 65.80% G GGC 78.60% S927 AGC G GGG 77.60% G GGT 79.30%I ATC 90.80% K AAG 70.70% V GTG 87.90% M930 ATG K AAG 82.30% R CGG73.80% R AGG 69.80% L1128 TTG A GCG 92.70% G GGG 89.70% K AAG 94.80% MATG 95.80% P CCG 98.40% R AGG 90.90% R CGG 88.50% S TCG 88.90% T ACG96.30% V GTG 93.90% W TGG 78.80% Y TAC 87.90%

Amino acid substitutions deemed beneficial to fatty alcohol productionwere next combined. PCR was used to amplify parts of the carBopt genecontaining various desired mutations, and the parts were joined togetherusing a PCR-based method (Horton, R. M., Hunt, H. D., Ho, S. N., Pullen,J. K. and Pease, L. R. 1989). The carBopt gene was screened without the60 bp N-terminal tag. The mutations combined in this combination libraryare shown in Table 9.

TABLE 9 CarB Mutations from the First Combination Library Mutation CodonE20V GTG E20S TCG E20R CGC V191S AGT F288R AGG F288S AGC F288G GGG Q473LCTG Q473W TGG Q473Y TAC Q473I ATC Q473H CAC A535S TCC

To facilitate screening, the resulting CarB combination library was thenintegrated into the chromosome of strain V668 at the lacZ locus. Thesequence of the carBopt gene at this locus is presented as SEQ ID NO:7.The genotype of strain V668 is MG1655 (ΔfadE::FRT ΔfhuA::FRTΔfabB::A329V ΔentD::T5-entD ΔinsH-11::P_(lacUV5) fab138 rph+ ilvG+) (asshown in Table 3 and FIG. 16). The strains were then transformed withplasmid pVA3, which contains TesA, a catalytically inactive CarB enzymeCarB[S693A] which destroys the phosphopantetheine attachment site, andother genes which increase the production of free fatty acids. Thecombination library was screened as described above for the error pronelibrary. V668 with integrated carB opt (A535S) in the lacZ region andcontaining pVA3 was used as the control. Hits were selected thatincreased the production of fatty alcohols and were subjected to furtherverification using shake-flask fermentations, as described in Example 5.The improved percentage of fatty alcohol production following shakeflask fermentation of recombinant host cells expressing CarB combinationmutants is shown in FIG. 12.

The integrated CarB combination mutants were amplified from theintegrated carB hits by PCR using the primers:

EG58 (SEQ ID NO: 33) 5′-GCACTCGACCGGAATTATCG; and EG626 (SEQ ID NO: 34)5′-GCACTACGCGTACTGTGAGCCAGAG.

These inserts were re-amplified using primers:

DG243 (SEQ ID NO: 35) 5′-GAGGAATAAACCATGACGAGCGATGTTCACGACGCGACCGACGGC;and DG210 (SEQ ID NO: 36) 5′-CTAAATCAGACCGAACTCGCGCAGG.

Using InFusion cloning, the pooled carB mutants were cloned into aproduction plasmid, pV869, which was PCR amplified using primers:

DG228 (SEQ ID NO: 37) 5′-CATGGTTTATTCCTCCTTATTTAATCGATAC; and DG318 (SEQID NO: 38) 5′-TGACCTGCGCGAGTTCGGTCTGATTTAG.

The carB mutant that performed the best in the shake-flask fermentationplasmid screen (carB2; Table 11) was designated VA101 and the controlstrain carrying carBopt [A535S] was designated VA82. See FIG. 13.

Amino acid substitutions in the reduction domain of carB deemedbeneficial to fatty alcohol production were combined with one of thebest carB-L combination library hits, “carB3” (Table 11). PCR was usedto amplify parts of the carBopt gene containing various desiredmutations in Reduction domain, and the parts were joined together usingSOE PCR. The mutations combined in this combination library are shown inTable 10.

TABLE 10 CarB Mutations from the Second Combination Library MutationCodon R827C TGC R827A GCA V926A GCG V926E GAG S927K AAG S927G GGG M930KAAG M930R AGG L1128W TGG

The combination library was screened as described above for the errorprone library. V668 with integrated carB3 in the lacZ region andcontaining pVA3 was used as a control. Hits were selected that exhibitedincreased production of fatty alcohols and were subjected to furtherverification using shake-flask fermentations, as described above. Theresults of a shake flask fermentation showing an improved percentage offatty alcohol production using a further CarB combination mutation(carB4) is shown in Table 11. A graphic depiction of the relativeconversion efficiency of low copy CarB variants is presented in FIG. 14.Results reported in Table 11 are from bioreactor runs carried out underidentical conditions.

TABLE 11 CAR Variants Name Mutation(s) Strain Tank data Notes carB None= WT (E20 V191 protein is SEQ ID NO: 7 F288 Q473) carB60 None + tag V324carB1 A535S V940 83% FALC; C12/C14 = 3.4 has one copy of 12H08chromosomal TE carB2 E20R, F288G, Q473I, A535S LH375 97% FALC; C12/C14 =3.6 has two copies of 12H08 chromosomal TE carB2 E20R, F288G, Q473I,A535S LH346 96% FALC; C12/C14 = 3.7 has one copy of 12H08 chromosomal TEcarB3 E20R, F288G, Q473H, A535S L combo library No examples run inbioreactors to date carB4 E20R, F288G, Q473H, A535S, R combo library 97%FALC; C12/C14 = 3.9 has two copies of 12H08 chromosomal TE R827A, S927G(VA-219) carA None See, U.S. Pat. Pub. protein is SEQ ID NO: 39 No.20100105963 FadD9 None See, U.S. Pat. Pub. protein is SEQ ID NO: 40 No.20100105963The DNA sequences of CarA, FadD9, CarB, and CarB60 are presented hereinas SEQ ID NO: 41, 42, 43 and 44, respectively.

Identification of Additional Beneficial Mutations in CarB Enzyme bySaturation Mutagenesis:

A dual-plasmid screening system was later developed and validated toidentify improved CarB variants over CarB4 for FALC production. Thedual-plasmid system met the following criteria: 1) Mutant clones producehigh FA titer to provide fatty acid flux in excess of CarB activity.This is accomplished by transforming a base strain (V668 with two copiesof chromosomal TE) with a plasmid (pLYC4, pCL1920_P_(TRC)_(_)carDead_tesA_alrAadp1_fabB[A329G]_fadR) that carries the FALC operonwith a catalytically inactive CarB enzyme CarB[S693A] to enhance theproduction of free fatty acids; 2) The screening plasmid with carBmutant template, preferably smaller than 9-kb, is amenable to saturationmutagenesis procedures and is compatible for expression with pLYC4; 3)The dynamic range of CarB activity is tunable. This is achieved bycombining a weaker promoter (P_(TRC1)) and alternative start codons (GTGor TTG) to tune CarB4 expression levels. 3) Good plasmid stability, atoxin/antitoxin module (ccdBA operon) was introduced to maintain plasmidstability.

Briefly, the screening plasmid pBZ1(pACYCDuet-1_P_(TRC1)-carB4GTG_rrnBter_ccdAB) was constructed from fourparts using In-Fusion HD cloning method (Clontech) by mixing equal molarratios of four parts (P_(TRC1), carB4 with ATG/TTG/GTG start codons,rrnB T1T2 terminators with ccdAB, and pACYCDuet-1 vector). The parts (1to 4) were PCR amplified by the following primer pairs:

(1) P_(TRC1) - Forward primer (SEQ ID NO: 45)5′CGGTTCTGGCAAATATTCTGAAATGAGCTGTTGACAATTAATCAAATC CGGCTCGTATAATGTGTG-3′and reverse primer (SEQ ID NO: 46) 5′-GGTTTATTCCTCCTTATTTAATCGATACAT-3′using pVA232 (pCL1920_P_(TRC)_carB4_tesA_alrAadp1_fabB[A329G]_fadR)plasmid as template. (1) carB4 with ATG/TTG/GTG start codons - Forwardprimer carB4 ATG (SEQ ID NO: 47)5′ATGTATCGATTAAATAAGGAGGAATAAACCATGGGCACGAGCGATGTT CACGACGCGAC-3′; carB4GTG (SEQ ID NO: 48) 5′ATGTATCGATTAAATAAGGAGGAATAAACCGTGGGCACGAGCGATGTTCACGACGCGAC-3′; and carB4 TTG (SEQ ID NO: 49)5′-ATGTATCGATTAAATAAGGAGGAATAAACCTTGGGCACGAGCGATGT TCACGACGCGAC-3′; andreverse primer carB4 rev (SEQ ID NO: 50)5′-TTCTAAATCAGACCGAACTCGCGCAG-3′, using pVA232 plasmid as template. (3)The rrnB T1T2 terminators with ccdAB - Forward primer rrnB T1T2 term(SEQ ID NO: 51) 5′-CTGCGCGAGTTCGGTCTGATTTAGAATTCCTCGAGGATGGTAGTGTG G-3′and reverse primer ccdAB rev (SEQ ID NO: 52)5′-CAGTCGACATACGAAACGGGAATGCGG-3′, using plasmid pAH008 (pV171_ccdBAoperon). (4) The pACYCDuet-1 vector backbone - Forward primer pACYCvector for (SEQ ID NO: 53)5′CCGCATTCCCGTTTCGTATGTCGACTGAAACCTCAGGCATTGAGAAGC ACACGGTC-3′ andreverse primer pACYC vector rev (SEQ ID NO: 54)5′-CTCATTTCAGAATATTTGCCAGAACCGTTAATTTCCTAATGCAGGA GTCGCATAAG-3′.

The pBZ1 plasmid was co-expressed with pLYC4 in the strain describedabove and validated by shake flask and deep-well plate fermentation. Thefermentation conditions were optimized such that CarB4_GTG templatereproducibly have ˜65% FALC conversion in both fermentation platforms asdescribed in Example 5. Results for shake flask fermentation are shownin FIG. 15.

Additional sites (18, 19, 22, 28, 80, 87, 90, 143, 212, 231, 259, 292,396, 416, 418, 530, 541, 574, 612, 636, 677, 712, 750, 799, 809, 810,870, 936, 985, 986, 1026, 1062, 1080, 1134, 1149, 1158, 1161, 1170)containing mutations in the improved CarB variants (Table 7) weresubjected to full saturation mutagenesis. Primers containing thedegenerate nucleotides NNK or NNS were used to mutate these positions toother amino acids by a PCR-based method (Sawano and Miyawaki 2000, Nucl.Acids Res. 28: e78). Saturation library was constructed using the pBZ1(pACYCDuet-1_P_(TRC1)-carB4GTG_rrnBter_ccdAB) plasmid template. Mutantclones were transformed into NEB Turbo (New England Biolab) cloningstrains and plasmids were isolated and pooled. The pooled plasmids werethen transformed into a V668 based strain carrying plasmid pLYC4 and thetransformants were selected on LB agar plates supplemented withantibiotics (100 mg/L spectinomycin and 34 mg/L chloramphenicol).

CarB variants from the saturation library were then screened for theproduction of fatty alcohols. Single colonies were picked directly into96-well plates according to a modified deep-well plate fermentationprotocol as described in Example 5. Hits were selected by choosingclones that produced a smaller total free fatty acid titer and a largertotal fatty alcohol titer compared to the control strain. To comparehits from different fermentation batches, the conversion of free fattyacids to fatty alcohols was normalized by calculating a normalized freefatty acid percentage. The NORM FFA (%) was also used in hits validationas described in Example 5. NORM FFA (%)=Mutant Percent FFA/ControlPercent FFA; where “Percent FFA” is the total free fatty acid speciestiter divided by the total fatty species titer. Hits were subjected tofurther validation using shake-flask fermentations as described inExample 5. The normalized free fatty acid (NORM FFA) column indicatesthe improvement in the enzyme, with lower values indicating the bestimprovement. “Hit ID” indicates the primary screening plate wellposition where the lower NORM FFA phenotype was found. Hits mutationswere identified by sequencing PCR products amplified from “Hit”containing pBZ1 plasmids using mutant carB gene-specific primers (BZ1for 5′-GGATCTCGACGCTCTCCCTT-3′ (SEQ ID NO:55) and BZ12_ccdAB uniqueprimer 5′-TCAAAAACGCCATTAACCTGATGTTCTG-3′ (SEQ ID NO:56). The NORM FFAvalues and mutations identified in validated hits are summarized inTable 12.

TABLE 12 Beneficial Mutations in CarB4 Enzyme identified During AminoAcid Saturation Mutagenesis WT Amino Hit ID Mutant Acid WT Codon (AminoAcid) Codon NORM FFA (%) D18 GAT P10H5(R) AGG 75.5 P6B4(L) CTG 83.6P4H11(T) ACG 80.8 P8D11(P) CCG 81.8 S22 AGC P1F3(R) AGG 57.7 P2G9(R) AGG55.7 P2A7(N) AAC 90 P8D7(G) GGG 82.1 L80 CTG P8H11(R) AGG 87.4 R87 CGTP7D7(G) GGG 85.2 P5D12(E) GAG 89.4 D750 GAT P8F11(A) GCG 87.6 I870 ATTP3A12(L) CTG 76.6

Identification of Novel Variants of CarB Enzyme by Full CombinatorialMutagenesis:

A full combinatorial library was constructed to include the followingamino acid residues: 18D, 18R, 22S, 22R, 473H, 473I, 827R, 827C, 870I,870L, 926V, 926A, 926E, 927S, 927K, 927G, 930M, 930K, 930R, 1128L, and1128W. Primers containing native and mutant codons at all positions weredesigned for library construction by a PCR-based method (Horton, R. M.,Hunt, H. D., Ho, S. N., Pullen, J. K. and Pease, L. R. 1989). Beneficialmutations conserved in CarB2, CarB3, and CarB4 (20R, 288G, and 535S)were not changed, therefore, carB2GTG cloned into pBZ1 (modifiedpBZ1_P_(TRC1) _(_)carB2GTG_ccdAB) was used as PCR template. Libraryconstruction was completed by assembling PCR fragments into CarB ORFscontaining the above combinatorial mutations. The mutant CarB ORFs werethen cloned into the pBZ1 backbone by In-Fusion method (Clontech). TheIn-Fusion product was precipitated and electroporated directly into thescreening strain carrying plasmid pLYC4. Library screening, deep-wellplate and shake flask fermentation were carried out as described inExample 5. The activities (NORM FFA normalized by CarB2, 100%) of CarBmutants with specific combinatorial mutations are summarized in Table13. CarB2, CarB4, and CarB5 (CarB4-S22R) are included as controls. TheNORM FFA column indicates the improvement in CarB enzyme, with lowervalues indicating the best improvement. The fold improvement (X-FIOC) ofcontrol (CarB2) is also shown. All mutations listed are relative to thepolypeptide sequence of CarB wt (SEQ ID NO:7). For example, CarB1 hasA535S mutation, and the CarBDead (a catalytically inactive CarB enzyme)carries S693A mutation which destroys the phosphopantetheine attachmentsite.

Novel CarB Variants for Improved Fatty Alcohol Production inBioreactors:

The purpose of identifying novel CarB variants listed in Table 13 is touse them for improved fatty alcohol production. The top CarB variant(P06B6-S3R, E20R, S22R, F288G, Q473H, A535S, R873S, S927G, M930R,L1128W) from Table 13 carries a spontaneous mutation (wild type AGC toAGA) at position 3. Both P06B6 CarB variants, namely CarB7 (amino acid Rby AGA at position 3-S3R, E20R, S22R, F288G, Q473H, A535S, R873S, S927G,M930R, L1128W), and CarB8 (wild type amino acid S by AGC at position3-E20R, S22R, F288G, Q473H, A535S, R873S, S927G, M930R, L1128W) weremade and cloned into the low copy number fatty alcohol productionplasmid backbone pCL1920 to generate the following fatty alcohol operonsdiffering only in CarB. The translation initiation codon (GTG) for allCarB variants (CarB2, CarB7, and carB8) was reverted to ATG to maximizeexpression.

-   -   pCL1920_P_(TRC) _(_)carB2_tesA_alrAadp1_fabB[A329G]_fadR    -   pCL1920_P_(TRC) _(_)carB7_tesA_alrAadp1_fabB[A329G]_fadR    -   pCL1920_P_(TRC) _(_)carB8_tesA_alrAadp1_fabB[A329G]_fadR

The above described plasmids were transformed into a V668 based strainwith one copy of chromosomal TE, and the resulted strains were screenedin bioreactors as described in EXAMPLE 4. The improvement (measured by %fatty alcohols in the bioreactor fermentation product) of CarB7 andCarB8 over CarB2 was shown in FIG. 16. The order of activity isCarB7>CarB8>CarB2. The position 3 mutation of CarB7 (AGC to an AGA Rrare codon) conferred higher activity than CarB8, in addition, SDS-PAGEanalysis of total soluble proteins revealed higher expression of CarB7than CarB8 and CarB2. The expression levels of CarB2 and CarB8 weresimilar. This is consistent with the CarB60 data described in EXAMPLE 6,both the position 3 AGA R rare codon mutation and the CarB60 tag at itsN-terminus can improve CarB expression. It is understood that the CarB7and CarB8 will perform better than CarB2 in strains with increased freefatty acids flux by either engineering the host strains and/orengineering the other components of the fatty alcohol production operon.

TABLE 13 Summary of CarB Variants Identified from Combinatorial Libraryin Dual-Plasmid system. Mutants NORM FFA (%) X-FIOC Mutations P06B6 16.56.06 S3R, E20R, S22R, F288G, Q473H, A535S, R873S, S927G, M930R, L1128WP13A3 23.9 4.18 D18R, E20R, S22R, F288G, Q473I, A535S, S927G, M930K,L1128W P02A2 26.5 3.77 E20R, S22R, F288G, Q473I, A535S, R827C, V926E,S927K, M930R P05H3 26.7 3.75 D18R, E20R, 288G, Q473I, A535S, R827C,V926E, M930K, L1128W P10F10 31.9 3.13 E20R, S22R, F288G, Q473H, A535S,R827C, V926A, S927K, M930R P01C12 34.2 2.92 E20R, S22R, F288G, Q473H,A535S, R827C P03B1 36.9 2.71 E20R, S22R, F288G, Q473I, A535S, R827C,M930R P06E4 36.9 2.71 E20R, S22R, F288G, Q473I, A535S, I870L, S927G,M930R P14C6 37.4 2.67 E20R, S22R, F288G, Q473I, A535S, I870L, S927GP05F10 40.4 2.48 D18R, E20R, S22R, F288G, Q473I, A535S, R827C, I870L,V926A, S927G P06C8 40.8 2.45 E20R, S22R, F288G, Q473H, A535S, R827C,I870L, L1128W P15E4 40.8 2.45 D18R, E20R, S22R, F288G, Q473H, A535S,R827C, I870L, S927G, L1128W P05H7 40.9 2.44 E20R, S22R, F288G, Q473I,A535S, R827C, I870L, S927G, L1128W P15A6 41 2.44 E20R, S22R, F288G,Q473I, A535S, R827C, I870L, S927G, M930K, L1128W P08F5 41.2 2.43 E20R,S22R, F288G, Q473H, A535S, I870L, S927G, M930K P14C7 41.3 2.42 E20R,F288G, Q473I, A535S, I870L, M930K P16H10 42.1 2.38 E20R, S22R, F288G,Q473H, A535S, S927G, M930K, L1128W PI6A1 44.1 2.27 D18R, E20R, S22R,F288G, Q473I, A535S, S927G, L1128W P14H4 44.2 2.26 E20R, S22R, F288G,Q473I, A535S, R827C, I870L, S927G PI5C1 46.5 2.15 D18R, E20R, S22R,F288G, Q473I, A535S, R827C, I870L, S927G, L1128W P16E5 47.2 2.12 D18R,E20R, S22R, F288G, Q473I, A535S, S927G, M930R, L1128W P15A3 47.2 2.12E20R, S22R, F288G, Q473H, A535S, V926E, S927G, M930R P05A2 52.4 1.91E20R, S22R, F288G, Q473H, A535S, R827C, I870L, V926A, L1128W CarB2 100 1E20R, F288G, Q473I, A535S CarB4 77.8 1.29 E20R, F288G, Q473H, A535S,R827A, S927G CarB5 48.9 2.04 E20R, S22R, F288G, Q473H, A535S, R827A,S927G CarB1 ND A535S CarB wt ND SEQ ID NO: 7 CarBDead ND S693A

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended merely to better illuminate thedisclosure and does not pose a limitation on the scope of the disclosureunless otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element as essential to thepractice of the disclosure. It is to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting. Preferred embodiments of thisdisclosure are described herein. Variations of those preferredembodiments may become apparent to those of ordinary skill in the artupon reading the foregoing description. The inventors expect skilledartisans to employ such variations as appropriate, and the inventorsintend for the disclosure to be practiced otherwise than as specificallydescribed herein. Accordingly, this disclosure includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the disclosure unless otherwise indicatedherein or otherwise clearly contradicted by context.

What is claimed is:
 1. A variant carboxylic acid reductase (CAR)polypeptide comprising an amino acid sequence having at least about 85%sequence identity to SEQ ID NO: 7, wherein the variant CAR polypeptideis genetically engineered to have at least one mutation at an amino acidposition selected from the group consisting of amino acid positions 3,18, 20, 22, 80, 87, 191, 288, 473, 535, 750, 827, 870, 873, 926, 927,930, and
 1128. 2. The variant CAR polypeptide of claim 1, whereinexpression of the variant CAR polypeptide in a recombinant host cellresults in a higher titer of fatty alcohol compositions compared to arecombinant host cell expressing a corresponding wild type polypeptide.3. The variant CAR polypeptide of claim 1, wherein the CAR polypeptideis a CarB polypeptide.
 4. The variant CAR polypeptide of claim 1,wherein the variant CAR polypeptide comprises a mutation selected fromthe group consisting of S3R, D18R, D18L, D18T, D18P, E20V, E20S, E20R,S22R, S22N, S22G, L80R, R87G, R87E, V191S, F288R, F288S, F288G, Q473L,Q473W, Q473Y, Q473I, Q473H, A535S, D750A, R827C, R827A, 1870L, R873S,V926A, V926E, S927K, S927G, M930K, M930R and L1128W.
 5. The variant CARpolypeptide of claim 4, wherein the variant CAR polypeptide comprisesmutation A535S.
 6. The variant CAR polypeptide of claim 4, wherein thevariant polypeptide comprises a combination of mutations selected fromthe group consisting of: E20R, F288G, Q473I, A535S; E20R, F288G, Q473H,A535S, R827A, S927G; E20R, S22R, F288G, Q473H, A535S, R827A, S927G; S3R,E20R, S22R, F288G, Q473H, A535S, R873S, S927G, M930R, L1128W; E20R,S22R, F288G, Q473H, A535S, R873S, S927G, M930R, L1128W; D18R, E20R,S22R, F288G, Q473I, A535S, S927G, M930K, L1128W; E20R, S22R, F288G,Q473I, A535S, R827C, V926E, S927K, M930R; D18R, E20R, 288G, Q473I,A535S, R827C, V926E, M930K, L1128W; E20R, S22R, F288G, Q473H, A535S,R827C, V926A, S927K, M930R; E20R, S22R, F288G, Q473H, A535S, R827C;E20R, S22R, F288G, Q473I, A535S, R827C, M930R; E20R, S22R, F288G, Q473I,A535S, I870L, S927G, M930R; E20R, S22R, F288G, Q473I, A535S, I870L,S927G; D18R, E20R, S22R, F288G, Q473I, A535S, R827C, I870L, V926A,S927G; E20R, S22R, F288G, Q473H, A535S, R827C, I870L, L1128W; D18R,E20R, S22R, F288G, Q473H, A535S, R827C, I870L, S927G, L1128W; E20R,S22R, F288G, Q473I, A535S, R827C, I870L, S927G, L1128W; E20R, S22R,F288G, Q473I, A535S, R827C, I870L, S927G, M930K, L1128W; E20R, S22R,F288G, Q473H, A535S, I870L, S927G, M930K; E20R, F288G, Q473I, A535S,I870L, M930K; E20R, S22R, F288G, Q473H, A535S, S927G, M930K, L1128W;D18R, E20R, S22R, F288G, Q473I, A535S, S927G, L1128W; E20R, S22R, F288G,Q473I, A535S, R827C, I870L, S927G; D18R, E20R, S22R, F288G, Q473I,A535S, R827C, I870L, S927G, L1128W; D18R, E20R, S22R, F288G, Q473I,A535S, S927G, M930R, L1128W; E20R, S22R, F288G, Q473H, A535S, V926E,S927G, M930R; and E20R, S22R, F288G, Q473H, A535S, R827C, I870L, V926A,L1128W.
 7. A recombinant host cell comprising an exogenouspolynucleotide sequence encoding a variant carboxylic acid reductase(CAR) polypeptide having at least 85% sequence identity to SEQ ID NO: 7and having at least one mutation at an amino acid position selected fromthe group consisting of position 3, 18, 20, 22, 80, 87, 191, 288, 473,535, 750, 827, 870, 873, 926, 927, 930, and 1128, wherein therecombinant host cell produces a fatty alcohol composition at a highertiter or yield than a host cell expressing a corresponding wild type CARpolypeptide when cultured in a medium containing a carbon source underconditions effective to express the variant CAR polypeptide.
 8. Therecombinant host cell of claim 7, wherein the SEQ ID NO: 7 is thecorresponding wild type CAR polypeptide.
 9. The recombinant host cell ofclaim 7, further comprising a polynucleotide encoding a) a thioesterasepolypeptide; b) a FabB polypeptide and a FadR polypeptide; or c) fattyaldehyde reductase (AlrA) polypeptide.
 10. The recombinant host cell ofclaim 7, wherein the host cell is selected from the group consisting ofa plant cell, an insect cell, a fungal cell, an algal cell, and abacterial cell.
 11. The recombinant host cell of claim 10, wherein thehost cell is a bacterial cell.
 12. The recombinant host cell of claim11, wherein the bacterial cell is an E. coli cell.
 13. A method ofmaking a fatty alcohol composition comprising culturing a recombinanthost cell comprising an exogenous polynucleotide sequence encoding avariant carboxylic acid reductase (CAR) polypeptide having at least 85%sequence identity to SEQ ID NO: 7 and having at least one mutation at anamino acid position selected from the group consisting of amino acidpositions 3, 18, 20, 22, 80, 87, 191, 288, 473, 535, 750, 827, 870, 873,926, 927, 930, and 1128, in a culture medium comprising a carbon sourceunder conditions suitable to produce the fatty alcohol composition,wherein the fatty alcohol composition is released from the host cellinto the culture medium.
 14. The method of claim 13, wherein the variantCAR polypeptide comprises a mutation selected from the group consistingof S3R, D18R, D18L, D18T, D18P, E20V, E20S, E20R, S22R, S22N, S22G,L80R, R87G, R87E, V191S, F288R, F288S, F288G, Q473L, Q473W, Q473Y,Q473I, Q473H, A535S, D750A, R827C, R827A, I870L, R873S, V926A, V926E,S927K, S927G, M930K, M930R, and L1128W.
 15. The method of claim 14,wherein the variant CAR polypeptide comprises mutation A535S.
 16. Themethod of claim 14, wherein the variant CAR polypeptide comprises acombination of mutations selected from the group consisting of: E20R,F288G, Q473I, A535S; E20R, F288G, Q473H, A535S, R827A, S927G; E20R,S22R, F288G, Q473H, A535S, R827A, S927G; S3R, E20R, S22R, F288G, Q473H,A535S, R873S, S927G, M930R, L1128W; E20R, S22R, F288G, Q473H, A535S,R873S, S927G, M930R, L1128W; D18R, E20R, S22R, F288G, Q473I, A535S,S927G, M930K, L1128W; E20R, S22R, F288G, Q473I, A535S, R827C, V926E,S927K, M930R; D18R, E20R, 288G, Q473I, A535S, R827C, V926E, M930K,L1128W; E20R, S22R, F288G, Q473H, A535S, R827C, V926A, S927K, M930R;E20R, S22R, F288G, Q473H, A535S, R827C; E20R, S22R, F288G, Q473I, A535S,R827C, M930R; E20R, S22R, F288G, Q473I, A535S, I870L, S927G, M930R;E20R, S22R, F288G, Q473I, A535S, I870L, S927G; D18R, E20R, S22R, F288G,Q473I, A535S, R827C, I870L, V926A, S927G; E20R, S22R, F288G, Q473H,A535S, R827C, I870L, L1128W; D18R, E20R, S22R, F288G, Q473H, A535S,R827C, I870L, S927G, L1128W; E20R, S22R, F288G, Q473I, A535S, R827C,I870L, S927G, L1128W; E20R, S22R, F288G, Q473I, A535S, R827C, I870L,S927G, M930K, L1128W; E20R, S22R, F288G, Q473H, A535S, I870L, S927G,M930K; E20R, F288G, Q473I, A535S, I870L, M930K; E20R, S22R, F288G,Q473H, A535S, S927G, M930K, L1128W; D18R, E20R, S22R, F288G, Q473I,A535S, S927G, L1128W; E20R, S22R, F288G, Q473I, A535S, R827C, I870L,S927G; D18R, E20R, S22R, F288G, Q473I, A535S, R827C, I870L, S927G,L1128W; D18R, E20R, S22R, F288G, Q473I, A535S, S927G, M930R, L1128W;E20R, S22R, F288G, Q473H, A535S, V926E, S927G, M930R; and E20R, S22R,F288G, Q473H, A535S, R827C, I870L, V926A, L1128W.
 17. The method ofclaim 13, wherein the host cell further comprises a polynucleotideencoding a) a thioesterase polypeptide; b) a FabB polypeptide and a FadRpolypeptide; or c) fatty aldehyde reductase (AlrA) polypeptide.
 18. Themethod of claim 13, wherein the host cell is selected from the groupconsisting of a plant cell, an insect cell, a fungal cell, an algalcell, and a bacterial cell.
 19. The method of claim 18, wherein the hostcell is a bacterial cell.
 20. The method of claim 19, wherein thebacterial cell is an E. coli cell.